Sustained release of nutrients in vivo

ABSTRACT

Nutritional compositions delivered in vivo in a time controlled manner sustainable over long periods of time, provide enhancing athletic performance, increased hand/eye coordination and concentration on the task at hand.

INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional patent applicationSer. No. 61/014,251 filed Dec. 17, 2007.

The foregoing application, and all documents cited therein or duringtheir prosecution (“appln cited documents”) and all documents cited orreferenced in the appln cited documents, and all documents cited orreferenced herein (“herein cited documents”), and all documents cited orreferenced in herein cited documents, together with any manufacturer'sinstructions, descriptions, product specifications, and product sheetsfor any products mentioned herein or in any document incorporated byreference herein, are hereby incorporated herein by reference, and maybe employed in the practice of the invention.

FIELD OF THE INVENTION

The invention relates to compositions for increasing performance ofathletes by providing appropriately timed release and increasedabsorption of nutrients including carbohydrates, amino acids, andelectrolytes.

BACKGROUND OF THE INVENTION

Contemporary understanding of sports related nutritional requirementsindicates that carbohydrate consumption plays a key role improvingathlete endurance and performance. With the entrance of Gatorade® andother similar sports drinks beginning in the mid 1960's more attentionhas been paid to understanding the role of various nutrients on humanperformance in athletic events.

Understanding the body's ability to absorb and process carbohydrates andother nutrients for maximal performance output has been studied by many.In 2003, the International Olympic Committee on Nutrition for Athletesissued a position stating that a high carbohydrate diet in the daysbefore competition helps enhance performance, particularly when exerciselasts longer than about 60 min and that athletes should aim to achievecarbohydrate intakes that meet the fuel requirements of their trainingprograms and also adequately replace their carbohydrate stores duringrecovery between training sessions and competition. However, this is adifficult task, especially for athletes in training who need a sustainedsupply of carbohydrates and other nutrients without the drawback of anintake of large amounts or smaller amounts but at more regularintervals, of food in order to meet the dietary requirements necessaryto maintain enhanced performance.

There is thus a need in the art to provide a sustained and controlledsupply of carbohydrates and other nutrients to individuals without thenecessity of intake of excess food and snacks.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

The present invention is based upon the premise that athleticperformance can be improved by providing controlled release ofnutritional supplements (such as carbohydrates and electrolytes).

The present invention relates to a composition comprising nutritionalsupplements (such as carbohydrates, amino acids, vitamins, and/orelectrolytes), which when administered to a human provides a sustainedrelease of the nutritional supplements over an extended period of time.Preferably, the nutritional supplements may be delivered in a sustainedand extended manner for peak athletic performance and recovery. Forinstance, the composition may release the nutritional supplements sothat absorption and oxidation rates of exogenous carbohydrates areincreased during exercise. According to one embodiment, the compositionreleases nutritional supplements such that saturation of the SGLT1 andGLUT-5 transporters with exogenous carbohydrates is maintained duringexercise.

In a preferred embodiment, a composition for in vivo consumption maycomprise nutritional supplements; and, compounds for sustained releaseof the nutritional supplements in vivo. Preferably, the nutritionalsupplements may comprise carbohydrates, amino acids, vitamins, andelectrolytes.

In another preferred embodiment, the compounds for the sustained releaseof the nutritional supplements may comprise one or more componentsselected from biodegradable polymers, bioadhesives, nanoparticles,colloidal suspensions and binders.

According to one embodiment, the biodegradable polymers and binders maybe selected from poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpolyorthoester, biodegradable polyurethanes, hydrogels, blends andcopolymers thereof.

In an advantageous embodiment, the invention encompasses nanoparticlesand microparticles comprising hydrogels. Advantageously, the hydrogelsmay comprise modified polysaccharides. The polysaccharides may comprisemodifications with chemical groups such as carboxylic acid (as incarboxymethyl cellulose [CAS no. 9000-11-7], starch glycolate [CAS no.9057-06-1], etc.), acryloyl or methacryloyl (as in starch acrylate [CASno. 39316-65-9], hydroxypropyl cellulose acrylate [CAS no. 94187-94-7],hydroxyethyl starch acrylate, etc.), hydroxyalkyl (as in hydroxypropylcellulose [CAS no. 9004-64-2], hydroxyethyl starch [CAS no. 9005-27-0],hydroxyethyl cellulose [CAS no. 9004-62-0], etc.), and ester (as instarch acetate, starch phosphate, etc.). The modified starch may alsocontain grafted fatty acid moieties (as in starch adipate [CAS no.39347-22-3], etc.), oligo(alkylene oxide) grafts (as in hydroxypropylcellulose, etc.), grafted hydrophobes such as cholesterol, graftedamphiphiles such as alkenylsuccinate (as in 1-octenylsuccinic acidanhydride modified starch, etc.) or side chain derivatizations tointroduce aldehydic or carboxylic groups.

In another advantageous embodiment, the invention may also comprisemethods of crosslinking of the microparticles and nanoparticles of thepresent invention to form hydrogels. Crosslinking may be performed usingfree radical initiators such as persulfate salts, or redox systemsinvolving ascorbic acid, or a naturally occurring crosslinker such asgenipin. Ionic crosslinking may also be contemplated, such as withanionic polysaccharides such as gellan.

In another yet advantageous embodiment, the invention may also comprisethe preparation of hydrogels. In an advantageous embodiment, a blend ofhydrophobically modified polysaccharide such as, but not limited to,hydroxypropyl cellulose and a carboxy containing polysaccharide such as,but not limited to, alginate or carboxymethyl cellulose may be used toprepare the hydrogel particles of the present invention. In anotheradvantageous embodiment, nanoparticle suspensions may be synthesized byself-assembly of chitosan and carboxymethyl cellulose hydrolysate,wherein the polymers may be hydrolyzed with the enzymes chitosanase andcellulase, respectively.

In another embodiment, hydrogels may be prepared from mixtures of acidicpolysaccharides such as, but not limited to, alginates, and basicpolysaccharides such as, but not limited to, oligosaccharide derivativesof chitosan; a basic polysaccharide such as, but not limited to,chitosan and anionic polysaccharide such as, but not limited to,hyaluronic acid; alginate and oxidized alginate blended with chitosan;grafted agar and sodium alginate blend with acrylamide; gellanco-crosslinked with scleroglucan; photocrosslinked modified dextran;starch reacted with glycidyl methacrylate; or polymerizable saccharidemonomers, such as sucrose, created by reaction of the sugar with epoxyacrylate, or methacryloyl chloride and acetyl chloride. The nanoparticleand microparticle hydrogels may be prepared using polymer self-assembly(temperature, pH, or ionic strength induced precipitation; micelleformation; colloidal precipitation because of electrostatic interactionsbetween oppositely charged polymers) or conventionalemulsification-based methods.

In another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto increase exercise duration and cumulative power output potential byat least about 1% to about 50%, about 50% to about 100%, about 100% toabout 500% or about 500% to about 1000% when compared to a control withno nutritional supplement ingestion pre and during exercise.

In another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto increase burst energy duration and power output by about 1% to about50%, about 50% to about 100%, about 100% to about 500% or about 500% toabout 1000% when compared to a control with no nutritional supplementingestion before and during exercise.

In another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto increase burst energy duration and power output by about 1% to about50%, about 50% to about 100%, about 100% to about 500% or about 500% toabout 1000% when compared to a control with no nutritional supplementingestion pre and during exercise.

In yet another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto increase the number of effective burst events by at least about 1% toabout 50%, about 50% to about 100%, about 100% to about 500% or about500% to about 1000% when compared to a control with no nutritionalsupplement ingestion pre and during exercise.

In another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto decrease the duration of recovery time between burst events by atleast about 1% to about 50%, about 50% to about 100%, about 100% toabout 500% or about 500% to about 1000% when compared to a control withno nutritional supplement ingestion before and during exercise.

In another preferred embodiment, the nutritional supplements arereleased in vivo in a sustained manner and in a concentration effectiveto increase continuous exercise power output (watts) by about 1% toabout 50%, about 50% to about 100%, about 100% to about 500% or about500% to about 1000%, advantageously about 50% at about VO₂ 62% for 90minutes and not less than about 25% increase in high intensity (burst)exercise, about VO₂ 86%, power output and duration following 90 min ofcontinuous exercise, and increase the number of effective “burst”periods by about 100% as compared to performance when utilizingcommercially available performance enhancing drinks.

In yet another preferred embodiment, the compounds for sustained releaseof the nutritional supplements in vivo release the nutritionalsupplements in effective amounts and concentrations over extendedperiods of time to increase exercise duration and cumulative poweroutput potential by about 1% to about 50%, about 50% to about 100%,about 100% to about 500% or about 500% to about 1000%, advantageously atleast about 10% to about 70%, increase burst energy duration and poweroutput by at least about 10% to about 70%, increase the number ofeffective burst events from about 10% to 100%, decreases the duration ofrecovery time between burst events by at least about 10% to about 100%,when compared to a control with no nutritional supplement ingestion preand during exercise.

In another preferred embodiment; the compounds for sustained release ofthe nutritional supplements in vivo release the nutritional supplementsin effective amounts and concentrations over extended periods of time todecrease duration of recovery time between burst events by at leastabout 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about50%, about 60%, about 70%, about 80%, about 90% or about 100% whencompared to a control with no nutritional supplement ingestion pre andduring exercise.

In another preferred embodiment, the compounds for sustained release ofthe nutritional supplements in vivo release the nutritional supplementsin effective amounts and concentrations over extended periods of time toincrease continuous exercise power output (watts) by about 2%, about 5%,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90% or about 100%, advantageously about 50% atabout VO₂ 62% for 90 minutes and not less than about a 25% increase inhigh intensity (Burst) exercise, about VO₂ 86%, power output andduration following 90 minutes of continuous exercise, and increase thenumber of effective burst periods by at least about 100% as compared toperformance when utilizing commercially available performance enhancingdrinks.

In another preferred embodiment, the compositions are formulated as abeverage, a gum, a food snack, a powder or any other consumable.

In another preferred embodiment, a method of improving athleticperformance and endurance comprises administering to an individual acomposition comprising nutritional supplements and, compounds forsustained release of the nutritional supplements in vivo, wherein thenutritional supplements comprise carbohydrates, amino acids, vitamins,and electrolytes; and, consuming the composition wherein the compoundsfor sustained release of the nutritional supplements in vivo release thenutritional supplements in effective amounts and concentrations overextended periods of time to increase exercise duration and cumulativepower output potential by at least about 1% to about 50%, about 50% toabout 100%, about 100% to about 500% or about 500% to about 1000% about10% to about 70%, increase burst energy duration and power output by atleast about 10% to about 70%, increase the number of effective burstevents from about 10% to 100%, decreases the duration of recovery timebetween burst events by at least about 10 to about 100%, when comparedto a control with no nutritional supplement ingestion before and duringexercise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1 depicts polysaccharides containing carboxylic acid groups;

FIG. 2 depicts an oxidized scleroglucan derivative;

FIG. 3 depicts Passerini and Ugi multicomponent condensation reactions;

FIG. 4 depicts the structure of genipin;

FIG. 5 depicts the structure of carrageenan;

FIG. 6 depicts vinyl-functionalized dextran;

FIG. 7 depicts glycidyl methacrylate adduct of starch resulting from (a)transesterification, and (b) ring opening of the epoxy group;

FIGS. 8A and 8B depict crosslinking of polysaccharides containinghydroxyl groups. P represents a polysaccharide segment;

FIG. 9 depicts a crosslinked hydroxypropyl cellulose macrogel and acolloidally stable dispersion of hydroxypropyl cellulose microgelparticles in water;

FIG. 10 depicts crosslinking of hydroxyl containing polysaccharidesusing TSTMP in the presence of sodium hydroxide; and

FIG. 11 depicts synthesis of acrylated hydroxypropyl cellulose.

DETAILED DESCRIPTION

While the making and using of the various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments described herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of the inventionor the scope of the claims appended hereto.

According to one embodiment, the composition of the present inventionprovides sustained release over extended periods of time ofcarbohydrates and optionally other nutrients to an individual.Preferably, carbohydrates are released from the composition in a mannerwhich maintains maximum carbohydrate absorption and oxidation rates fora prolonged period of time. Generally, the maximal carbohydrateabsorption rate in an individual is in a range of about 1.2 to about 1.7g/min. A peak carbohydrate oxidation rate of about 1.75 g/min can beachieved, for example, when carbohydrates are administered as a blend ofglucose (1.2 g/min) and fructose (0.8 g/min). See, e.g., Azevedo, J. L.Jr.; Tietz, E.; Two-Feathers, T.; Paull, J.; Chapman, K. Lactate,Fructose and Glucose Oxidation Profiles in Sports Drinks and the Effecton Exercise Performance. PLoS ONE [Online] 2007, 2, pp e927-e927(doi:10.1371/journal.pone.0000927), for representative carbohydrateoxidation profiles.

The rate and extent of exogenous carbohydrate absorption may be limitednot only by the amount of carbohydrate available but also by the maximumintestinal transport capacity for glucose and fructose. Intestinaltransport of glucose is mediated by a sodium dependent glucosetransporter (SGLT1), located in the brush-border membrane. SGLT1transporters may become saturated at a glucose ingestion rate of ˜1g/min. Fructose on the other hand is absorbed from the intestine byGLUT-5, a sodium-independent facilitative fructose transporter.Generally, ingestion of a mixture of carbohydrates that have differenttransport mechanisms for absorption in to the blood stream,simultaneously increases carbohydrate and water absorption.

Applicants have approximated the number of calories required based oncertain activity levels as indicated in Table 1. The energy requirementis estimated based on the weight-normalized Caloric burn rate (kcal perminute of activity per unit body mass of an individual) and the Caloriccontent of glucose (˜4 kcal/g). The energy available from exogenousglucose oxidation is about 396 kcal. As can be seen from Table 1, thisenergy is insufficient to meet the total energy requirement of thelisted activities. The present invention seeks to narrow the Caloric gapusing a controlled-release nutrient formulation.

TABLE 1 Energy requirement based on activity levels Outside CompetitiveRunning Running construction soccer (8.5 min/mile) (12 min/mile) workCaloric burn rate (kcal min⁻¹ lb⁻¹) 0.0757 0.0871 0.0606 0.0417 Totalenergy required* (kcal) 1022 1176 818 563 Required glucose oxidationrate^(§) (g 0.0189 0.0218 0.0151 0.0104 min⁻¹ lb⁻¹) Total glucoserequired* (g) 256 294 204 141 Exogenous glucose oxidized^(#) (g) 99 9999 99 Difference (g) 157 195 105 42 Energy obtained from exogenous 396396 396 396 glucose (kcal) Caloric gap (kcal) 627 780 422 167 *Based ona 150-lb individual and a 90-min activity. ^(§)Oxidation rate of glucosethat is required to sustain the Caloric burn rate reported in row 1. Thevalues are estimated using the fact that 1 g of pure carbohydrate yieldsabout 4 kcal of energy. ^(#)Based on an exogenous glucose oxidation rateof ~1.1 g/min, which is determined by the rate of transport of glucose(from the intestinal lumen in to the blood stream) across the intestinalepithelium.

The term “sustained release” (i.e., extended release and/or controlledrelease) are used herein to refer to nutritional supplement, for examplecarbohydrates etc., delivery system or composition that is introducedinto the body of a human and that continuously releases a stream of oneor more nutrients over a predetermined time period and at a levelsufficient to achieve a desired effect throughout the predetermined timeperiod. Reference to a continuous release stream is intended toencompass release that occurs as the result of diffusion-limited releaseof the component from the matrix, or biodegradation in vivo of thecomposition, or a matrix or component thereof, or as the result ofmetabolic transformation or dissolution of the added nutrients or otherdesired agent(s). Delayed release may be achieved by entrapping thenutrients within particulate carriers with mucoadhesive surfacecharacteristics. Adhesion of the nutrient-loaded particles to intestinalmucosa will increase retention time of the particles inside theintestinal lumen, thereby providing continuous release and transport ofnutrients across the epithelium into blood, beyond the normal retentiontime of non-adhesive composition inside the gastrointestinal tract.

In a preferred embodiment, the nutritional supplement composition is inthe form of a solution, suspension, gel capsule, powder, snack (e.g. abar), granola form, or tablet. The “delivery” of nutrients comprises,for example, suspending the nutrients individually or in combinations insustained release particulate microparticles; compounds which bind tothe nutrients with different affinities and the like. According to oneembodiment, the requisite volume for consumption by the individual isabout 500 mL when in liquid form, however, formulations increasing andor decreasing the concentrations and amounts are contemplated.

Examples of nutrients include, but not limited to carbohydrates,proteins, amino acids, vitamins, co-enzymes, phospholipids, minerals,and electrolytes. Examples of vitamins and co-enzymes that may bedelivered using this invention include but are not limited to water orfat soluble vitamins such as thiamin, riboflavin, nicotinic acid,pyridoxine, pantothenic acid, biotin, flavin, choline, inositol andparaminobenzoic acid, carnitine, vitamin C, vitamin D and its analogs(such as ergocalciferol, calcitriol, doxercalciferol, and paricalcitol),vitamin A and the carotenoids, retinoic acid, vitamin E and vitamin K.

In a preferred embodiment, the nutritional composition comprisescarbohydrates that are taken up by different receptors, e.g. SGLT andGLUT receptors. Suitable carbohydrates include, but are not limited to,mono-, di- and polysaccharides such as glucose, sucrose, maltose as wellas more complex edible carbohydrates such as maltodextrins. According toone preferred embodiment, the composition includes a blend of glucoseand fructose. The weight ratio of glucose to fructose preferably rangesfrom about 1:1 to about 100:1, about 5:1 to about 95:1, about 10:1 toabout 90:1, about 15:1 to about 85:1, about 20:1 to about 80:1, about25:1 to about 75:1, about 30:1 to about 70:1, about 35:1 to about 65:1,about 40:1 to about 60:1, about 45:1 to about 55:1 or about 50:1.Preferably, the composition includes from about 0.1 to about 99.9 wt. %,about 1 to about 99 wt. %, about 5 to about 95 wt. %, about 10 to about90 wt. %, about 15 to about 85 wt. %, about 20 to about 80 wt. %, about25 to about 75 wt. %, about 30 to about 70 wt. %, of carbohydrates,about 35 to about 65 wt. %, about 40 to about 60 wt. %, about 45 toabout 55 wt. %, or about 50 wt. %, calculated on a 100% dry matter basisof the composition.

In another preferred embodiment, the composition includes amino acids.The amino acids may be in the form of free amino acids or peptides, andare preferably present in an amount in the range of from about 0.1 toabout 99.9 wt. %, about 1 to about 99 wt. %, about 5 to about 95 wt. %,about 10 to about 90 wt. %, about 15 to about 85 wt. %, about 20 toabout 80 wt. %, about 25 to about 75 wt. %, about 30 to about 70 wt. %,of carbohydrates, about 35 to about 65 wt. %, about 40 to about 60 wt.%, about 45 to about 55 wt. %, or about 50 wt. % calculated on a 100%dry matter basis of the composition.

The peptide material can be derived from proteins of animal or plantorigin and examples of such proteins are milk proteins, meat proteins,soy proteins, wheat proteins, pea proteins, rice proteins and maizeproteins. Preferably the protein raw material is wheat gluten protein ora subfraction thereof such as gliadin. In the present context, the term“peptide material” is understood to indicate a protein hydrolysate andmay contain all types of peptides that may vary in length as well as acertain amount of free amino acids resulting from the hydrolysis. Theprotein raw material is hydrolyzed by one or more hydrolytic enzymes.The hydrolytic enzyme can be of animal, plant, yeast, bacterial orfungal origin. Preferably enzyme preparations are used which have a lowexo-peptidase activity to minimize the liberation of free amino acidsand to improve taste profiles of the protein hydrolysates. The preferredhydrolyzed protein material of the present invention has an averagepeptide chain length in the range of 1-40 amino acid residues and morepreferably in the range of 1-20 amino acid residues. The average peptidechain can be determined using the method as described in WO 96/26266.Further the peptide material is present in an amount of about 0.1-90 wt.%, calculated on dry matter basis of the composition.

Other optional components of the composition according to the inventionare vitamins, minerals, electrolytes, flavors, antioxidants, componentshaving co-enzyme and antioxidant properties, lipids includingemulsifiers, and proteins for meeting specific nutritional and/orphysiological needs.

In another preferred embodiment, the nutritional composition comprises abinder and/or biodegradable polymer or mixture of biodegradable polymerswith appropriate time release characteristics and release kinetics. Thecomposition of the invention may then be formed into a microparticlescomprising the appropriate nutritional compositions suitable forproviding efficacious concentrations of the compounds of the inventionover a prolonged period of time without the need for frequent re-dosing.The composition of the present invention can be incorporated into thebiodegradable polymer or polymer mixture in any suitable manner known toone of ordinary skill in the art and may form a homogeneous matrix withthe biodegradable polymer.

In another preferred embodiment, the nutrient supplements are present innano suspensions/colloidal particles. The nanoparticles or colloidalparticles (CP) can form a stable colloidal suspension in water and in aphysiological medium. The CP associate with the nutrients, e.g.carbohydrates, in aqueous media by a spontaneous mechanism; and the CPrelease the nutrients in a physiological medium and, more precisely, invivo. The release kinetics depend on the nature of the polymer that isthe CP precursor. A protein, whose nutritional value depends on thetertiary structure of the molecule may also be delivered by this method,using biocompatible polymer hosts that will not denature the protein.

Thus, by varying the specific structure of the polymers, it is possibleto control the association and release phenomena from the kinetic andquantitative points of view.

Another preferred embodiment of the invention concerns the preparationof: selected particles; and other selected particles which arestructured, submicron and capable of being used especially for carryingone or more nutrients, these particles being individualized (discrete)supramolecular arrangements that are: based on linear amphiphilicpolyamino acids having peptide linkages and comprising at least twodifferent types of hydrophilic repeating amino acids, and hydrophobicrepeating amino acids, the amino acids of each type being identical toor different from one another; capable of associating at least onenutrient in colloidal suspension, in the undissolved state, andreleasing it, especially in vivo in a prolonged and/or delayed manner;and stable in the aqueous phase at a pH of between 4 and 13, in theabsence of surfactant(s).

Preferably, the particles are submicron structured particles capable ofbeing used especially for carrying one or more nutrients, theseparticles being discrete supramolecular arrangements; capable ofassociating at least one nutrient in colloidal suspension, in theundissolved state, and releasing it, especially in vivo, in a prolongedand/or delayed manner; and stable in the aqueous phase at a pH ofbetween 4 and 13, in the absence of surfactant(s).

In another preferred embodiment, the composition can be formulated toencapsulate the nutritional compositions in microspheres ormicroparticles so that it may be admixed or formulated into any form,such as a powder, a beverage, gum, nutritional food product, pill andthe like.

A “microsphere” or “microparticle”, as defined herein, includes aparticle of a biocompatible solid-phase material having a diameter ofabout one millimeter to about one micrometer, or less, wherein theparticle may contain a biologically active agent and, wherein thesolid-phase material sustains the in vivo release of the nutritionalcompositions from the microsphere. A microsphere can have a spherical,non-spherical or irregular shape. The typical microsphere shape isgenerally spherical.

A “biocompatible” material, as defined herein; means that the material,and any degradation products of the material, is non-toxic to therecipient and also presents no significant deleterious or untowardeffects on the recipient's body.

In a preferred embodiment, the microspheres contain a mixture ofnutritional compounds and the microsphere is composed of a biodegradablematerial that is released over a certain period of time. For example, inorder to provide an initial burst of nutrients to provide an immediatereservoir of energy or nutrients to the individual, the nutritionalcompounds are formulated as such and can contain a variety ofcarbohydrates, amino acids, electrolytes, vitamins, etc. in differingratios. The second group can contain a differing ratio ofcarbohydrates:amino acids:vitamins etc., or strictly different orsimilar carbohydrates that are released over a longer period of time tomaintain a sustainable release of the nutrients. The formulation of thenutrients in the microspheres and the timing of release can be varieddepending on the types of activity, the individual, age, weight andnutritional needs. For example, a marathon runner (sustained nutritionover long period) would have different nutritional needs to a sprinter(burst of nutrition).

In another preferred embodiment, different types of carbohydrates, e.g.those that are taken up by for example, SGLT transporters versus GLUTtransporters are added in differing ratios at differing release rates toachieve the results as described infra.

In another preferred embodiment, compositions comprise compounds whichdissolve over a period of time in vivo sequentially in acid, neutral andweak alkaline regions of the gastrointestinal tract. These compoundsinclude for example, an acidic polymeric dispersion coating as the firstcoating to prolong nutrient supplement release. In this embodiment, themicroparticle comprises as a core a material comprising calciumcarbonate, sugar, dextrose and nonpareil seeds. The first coating is amaterial which retards rapid passage of water. The first coating ispreferably an aqueous dispersion of poly(methacrylic acid-co-ethylacrylate) (commercially available under the designation EudragitL30D-55). The second coat is a latex acrylic polymer. The second coatingis preferably poly(ethyl acrylate-co-methylmethacrylate-co-2-trimethylammonioethyl methacrylate chloride)(commercially available under the designation Eudragit RS-30D). Thethickness of the second coating is established to achieve the desiredtime-release rate for the drug.

The time release products are preferably substantially spherical inconfiguration. The diameter of the time release drug products typicallyranges between 20 and 650 microns, between 30 and 500 microns or between40 and 350 microns and is preferably between about 50 and 250 micronswhen the products are in a liquid suspension form. It is a feature ofthe present invention that the time release nutrient compositioncontaining products of the present invention, because of their size, canbe suspended in an aqueous medium, thereby providing a liquidsuspension.

In this embodiment, the nutrient compositions are formulated as a timerelease formulation comprising: a core which can be optional;nutritional supplements bound to the core; a first coating havinglimited permeability to water; and a second coating, which is morepermeable to water than the first coating, wherein the first and secondcoatings together comprise the time release components of the nutrientcompositions.

The core will generally have a diameter of about 19 to 57, about 20 to56, about 21 to 55, about 22 to 54, about 23 to 53, about 24 to 52,about 25 to 51, about 26 to 50, about 27 to 49, about 28 to 48, about 29to 47, about 30 to 46, or about 31 to 45 microns. The core is generallycomprised of an inert ingredient, preferably a material selected fromthe group consisting of calcium carbonate, sugar, dextrose and nonpareilseeds.

The first coating, which has a limited permeability to water and whichretards rapid passage of acid and water. This first coating willtypically have a diameter of between about 1.30 and 4.60, about 1.40 and4.50, about 1.50 and 4.40, about 1.60 and 4.30, about 1.70 and 4.20,about 1.80 and 4.10, about 1.90 and 4.00, about 2.00 and 3.90, about2.10 and 3.80, about 2.20 and 3.70, about 2.30 and 3.60 or about 2.40and 3.50 microns. The first coating is preferably an acidic polymericdispersion coating which prolongs drug release, more preferably anaqueous dispersion of poly(methacrylic acid-co-ethyl acrylate). Such apolymer is commercially available under the name EUDRAGIT L30D-55. Thecore and first coating together typically have a diameter of betweenabout 60 and 77, about 61 and 76, about 62 and 75, about 63 and 74,about 64 and 75 or about 65 and 74 microns.

It is appreciated that the first and second coatings together comprisethe time release components of the product of the present invention. Thefirst and second coatings together effect time release of the orallyadministrable drug within an individual over a maximum period of about12 hours. It is appreciated by those skilled in the art that thethickness of the second coating can be altered to achieve the desiredtime release rate for the supplement. That is, the thickness of thesecond coating can be increased to achieve a longer period of timerelease in the body. The coatings work due to differential porosity. Theinner coating comprised of, for example, poly(methacrylic acid-co-ethylacrylate) is sensitive to pH. Nutrient transport across the innercoating is determined by the porosity and water content of the coating,both of which are determined by the different pH values within regionsof the gastrointestinal tract. In an acidic environment (in thestomach), the inner coating becomes relatively hydrophobic and shrinks,leading to decreased pore size and nutrient permeability. In contrast,the pH inside the intestinal lumen is higher. The inner coating becomesrelatively hydrophilic due to ionization, and allows faster release ofnutrients from the particle cores. The outer coating is notpH-responsive, but can be used to control nutrient permeability bycontrolling the pore size. The present invention provides in the firstand second coating porosity such that water entering the time releasecomponent will pass through the second coating more rapidly that throughthe first coating and the drug and water exiting the time-releasecomponent will pass through the first coating more slowly than throughthe second coating. In the preferred form, passage through each coatingis by mechanical means with the passage through the first coating beingaugmented by ionic interaction.

In another preferred embodiment, one or more of nutrient supplements arebound or encapsulated by a particle which is stable in an aqueousenvironment and are released over an extended period of time once thesupplements have been consumed.

The composition according to the invention may have the form of apowder, gum, a beverage or any other food product. A beverage accordingto the invention can be prepared by dissolving the above-definedingredients in an appropriate amount of water. Preferably an isotonicdrink has been prepared. For drinks; intended to be used during andafter exercise it is recommended to have a concentration of thecomposition according to the invention in the range of about 0.10-60 wt.% calculated on the total weight of the drink.

In a preferred embodiment, the formulation has a viscosity and“mouth-feel” approximately equivalent to water. The viscosity of theformulation can be quantified using a capillary viscometer, such as theUbblehold viscometer, by determining the time required for the liquid tofall from one fiducial mark to another in a glass capillary [See, e.g.,Pearce, E. M.; Wright, C. E.; Bordoloi, B. K. Laboratory Experiments inPolymer Synthesis and Characterization; Pennsylvania State University:University Park, 1982; p. 187]. Room temperature viscosity of water isabout 1 cP, while that of olive oil is about 80 cP, castor oil about1000 cP and corn syrup about 1400 cP. The viscosity of fat-free milk isabout 30 cP [Vesa, T. H.; Marteau, P. R.; Briet, F. B. et al. Am. J.Clin. Nutr. 1997, 66, 123-126].

In another preferred embodiment; the composition comprises flavoringagents which provide a variety of tastes/aromas that are pleasant,palatable and impart a feeling of confidence for adults, adolescents,and child consumers.

In one preferred embodiment, consumption of the nutrient compositionsincreases exercise duration and cumulative power output potential by atleast about 2%, about 5%, about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90% or about 100% whencompared to no supplemental carbohydrate (i.e. the nutrientcompositions) ingestion pre and during exercise.

In another preferred embodiment, consumption of the nutrientcompositions increases burst energy duration and power output by atleast about 2%, about 5%, about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90% or about 100% whencompared to no supplemental carbohydrate ingestion pre and duringexercise.

In another preferred embodiment, consumption of the nutrientcompositions increases the number of effective burst events by at leastabout 1% to about 50%, about 50% to about 100%, about 100% to about 500%or about 500% to about 1000% when compared to no supplementalcarbohydrate (i.e. the nutrient compositions) ingestion pre and duringexercise.

In another preferred embodiment, consumption of the nutrientcompositions decreases the duration of recovery time between burstevents by at least about 2%, about 5%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% orabout 100% when compared to no supplemental carbohydrate (i.e. thenutrient compositions) ingestion pre and during exercise.

In another preferred embodiment, consumption of the nutrientcompositions results in about 1% to about 50%, about 50% to about 100%,about 100% to about 500% or about 500% to about 1000% increase incontinuous exercise power output (watts) at about VO₂ 62% for 90 minutesand not less than about a 25% increase in high intensity (Burst)exercise, about VO₂ 86%, power output and duration following 90 minutesof continuous exercise, and increase the number of effective “burst”periods by >100% as compared to performance when utilizing asperformance enhancing drinks such as GATORADE, CYTOMAX, or POWERADE.

The measurement of cumulative power output potential is well known toone of skill in the art (see, e.g., Byrne, C.; Twist, C.; Eston, R.Neuromuscular function after exercise-induced muscle damage: theoreticaland applied implications. Sports Med. 2004, 34, 49-69; Hunter, A.; St,C.; Lambert, M. et al. Effects of supramaximal exercise on theelectromyographic signal. Br. J. Sports Med. 2003, 37, 296-299; andWilliams, S. G.; Cooke, G. A.; Wright, D. J. et al. Peak exercisecardiac power output: A direct indicator of cardiac function stronglypredictive of prognosis in chronic heart failure. Eur. Heart J. 2001,22, 1496-1503). In a particularly advantageous embodiment, theyo-yo-intermittent recovery test is utilized for measuring cumulativepower output potential (see, e.g., Krustrup, P.; Mohr, M.; Amstrup, T.R. et al. The yo-yo intermittent recovery test: physiological response,reliability, and validity. Med. Sci. Sports Exerc. 2003, 35, 697-705).

In another preferred embodiment, consumption of the nutrientcompositions results in an increased sustainability in concentration,hand/eye coordination etc. during extended exercise. Tests of thisnature are known in the art and anyone or more can be utilized. See, forexample, U.S. Pat. No. 7,300,365. This would translate into improvedathletic performance but also aid promotion for non-athletic, academicand or work, performance uses.

In another preferred embodiment, consumption of the nutrientcompositions results in improved concentration and brain function ascompared to no supplemental carbohydrate (i.e. the nutrientcompositions) ingestion.

In another preferred embodiment, consumption of the nutrientcompositions is expected to result in improved and stabilized bloodglucose concentration and is therefore a beneficial beverage or foodalternative for individuals with diabetes.

In another preferred embodiment, the compositions are packaged foraccessibility during longer periods of athletic exertion (e.g., amarathon) or when immediate energy and associated supplements arerequired, the product comprising the nutrients is in a solid or gel fromwhich is wearable, yet protected, on the person in a ready to consumeform.

In another preferred embodiment, the nutritional composition is packagedso that it is easily handled and stored in backpacks, duffel bags,pockets, etc. Preferably, the packaging is environmentally friendly.

In a preferred embodiment the nutritional compositions are admixed witha biodegradable binder or encapsulated within a biodegradablemicrosphere which allows for sustained release of desired carbohydratesand other nutrients. “Biodegradable”, as defined herein, means thepolymer will degrade or erode in vivo to form smaller chemical species.Degradation can result, for example, by enzymatic, chemical and/orphysical processes. Suitable biocompatible, biodegradable polymersinclude, for example, polysaccharides, poly(lactide)s, poly(glycolide)s,poly(lactide-co-glycolide)s, poly(lactic acid)s, poly(glycolic acid)s,poly(lactic acid-co-glycolic acid)s, polycaprolactone, polycarbonates,polyesteramides, polyanhydrides, poly(amino acids), polyorthoesters,polyacetyls, polycyanoacrylates, polyetheresters, poly(dioxanone)s,poly(alkylene alkylate)s, copolymers of polyethylene glycol andpolyorthoester, biodegradable polyurethanes, hydrogels, blends andcopolymers thereof.

Biocompatible, non-biodegradable polymers suitable for the methods andcompositions of the present invention include non-biodegradable polymersselected from the group consisting of polyacrylates, polymethacrylates,polymers of ethylene-vinyl acetates and other acyl substituted celluloseacetates, non-degradable polyurethanes, polystyrenes, polyvinylchloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonatepolyolefins, polyethylene oxide, hydrogels, blends and copolymersthereof.

In another preferred embodiment, hydrogels are used in the sustainedrelease of the nutritional supplements. Physical polymeric hydrogelshave been widely explored for biomaterials applications. Examplesinclude hydrogels formed by complexation of enantiomeric polymer orpolypeptide segments and hydrogels with temperature- or pH-sensitiveproperties. They attract special attention for sustained drug deliverybecause of the mild and aqueous conditions involved in trapping delicatebioactive agents such as proteins. For example, in situ formedhydrogels, formed from thermosensitive block copolymers, have also beenproposed as sustained release matrices for drugs. They have theadvantage that there is no chemical reaction involved in the gelformation. These copolymer hydrogels are usually designed formacromolecular drugs such as proteins and hormones. Preferably thepolymer is in an aqueous solution, which forms a hydrogel. For example,suitable aqueous polymer solutions contain about 1% to about 80%, about2% to about 75%, about 3% to about 70%, about 4% to about 65%, about 3%to about 70%, about 4% to about 65%, about 5% to about 60%, about 6% toabout 55%, about 7% to about 50%, about 8% to about 45%, about 9% toabout 42% polymer, preferably about 10% to about 40% polymer. Suitablehydrogels can also contain about 1% to about 20%, about 2% to about 19%,about 3% to about 18%, about 4% to about 17% cyclodextrin (w/w) (basedon the weight of total solution), preferably about 5% to 15%cyclodextrin, to solubilize nutrients that have limited watersolubility. The hydrogel is typically formed using an aqueous carrierfluid. For example, typical aqueous solutions contain about 1% to about80%, about 2% to about 75%, about 3% to about 70%, about 4% to about65%, about 3% to about 70%, about 4% to about 65%, about 5% to about60%, about 6% to about 55%, about 7% to about 50%, about 8% to about45%, about 9% to about 42% polymer, preferably about 10% to about 40%polymer.

The hydrogel composition may also contain a secondary polymer, which maycomplex with the nutrient, conjugate the nutrient, or both. Thesecondary polymer may suitably be a polyester, polyurethane, polyamide,polyether, polysaccharide, poly(amino acid), polypeptide, or a protein.Preferably the secondary polymer is a di- or mono-functional polymer orpolyionic polymer with poly(ethylene glycol) segments. In the case wherenutritional supplements conjugate or complex to the hydrogels, then thehydrogel formulations act not only as a matrix but also a carrier of thenutritional supplements. This means that the nutritional supplements,e.g. a variety of carbohydrates, are not only physically entrapped inthe hydrogel, but also are complexed or conjugated to the molecules thatform the hydrogel. A secondary polymer may also be used to alter theproperties, such as porosity and viscosity, of the hydrogel matrix.

The properties of the hydrogels are tunable by using different polymerblock molecular weights, by adjusting the cyclodextrin content, andthrough the use of secondary polymers. For example, the hydrogel may beadjusted to be a more flexible hydrogel or a more rigid hydrogel. Thehydrogel structure can be tailored to have variable viscosity and longeror shorter drug release rates. The degree of hydrophobicity of thepoly(hydroxyalkanoate) can also be selected for a desired sustainedrelease rate.

The duration of extended release is dependent on the molecular weightsof the block polymers, particularly the molecular weight of thehydrophobic poly(hydroxyalkanoate) section (e.g., PHB). The release ratemay be altered in accordance with the invention to achieve a desiredduration of response by selecting: a particular poly(hydroxyalkanoate);the stereo-isomeric state of the selected poly(hydroxyalkanoate); themolecular weight of the selected poly(hydroxyalkanoate); and therelative quantity of cyclodextrin used in the hydrogel, to achieve adesired duration and rate of sustained release. The molecular weight andselection of the hydrophilic poly(alkylene oxide) also impacts thesustained release kinetics, but to a lesser extent than the hydrophobicpoly(hydroxyalkanoate) component. Secondary polymers may also beutilized to change the release kinetics. Hydrogels can provide sustainedrelease over a period of one or more days by adjustment of the molecularweights of the block polymers and the copolymer, as well as thecyclodextrin content within the hydrogel of the present invention andthe potential use of secondary polymers.

Microencapsulation of components of the nutritional supplement inbiodegradable polymers such as polylactide-polyglycolide is alsocontemplated. Depending on the ratio of component to polymer, and thenature of the particular polymer employed, the rate of component releasemay be sustained. Examples of other biodegradable polymers includepoly(orthoester)s and poly(anhydride)s. The formulations are alsoprepared by entrapping the component in liposomes or microemulsionswhich are compatible with body tissue.

Further, the terminal functionalities of a polymer can be modified. Forexample, polyesters may be blocked, unblocked or a blend of blocked andunblocked polymers. A blocked polyester is as classically defined in theart, specifically having blocked carboxyl end groups. Generally, theblocking group is derived from the initiator of the polymerization andis typically an alkyl group. An unblocked polyester is as classicallydefined in the art, specifically having free carboxyl end groups.

In an advantageous embodiment, blends of polysaccharides are utilized tosynthesize aqueous dispersions of microparticles or nanonparticles.Advantageously, the polysaccharides are hydrophobically modifiedpolysaccharides wherein the polysaccharides form interpenetratingpolymer networks. In an especially advantageous embodiment, thepolysaccharides contain carboxylic acid groups, such as, but not limitedto, the polysaccharides of FIG. 1.

Without being bound by theory, it is expected that the carboxycontaining hydrogel particles are in a collapsed state in the acidicenvironment of the stomach. Hence, the encapsulated sugar molecules areretained within the particles in the stomach. The hydrogel particleswill achieve an expanded state when they reach the small intestine (pH5-7), and will release the encapsulated sugar at a rate faster than thatin the stomach. A key feature of the proposed polysaccharide hydrogelsis their pH responsiveness. Ideally, the hydrogels should not swell inthe acidic environment of the stomach, but should swell upon entry intothe small intestine and release the encapsulated sugars at a controlledrate. Advantageously, the carbohydrates of the present invention arecontrolled release particles dispersed in an aqueous medium, but mayalso be stored in a solid particulate form.

In a particularly advantageous embodiment, the hydrogels comprisehydrophobized polysaccharides. Polysaccharides may be functionalizedwith hydrophobes such as cholesterol. For example, polysaccharides suchas, but not limited to, pullulan, dextran and mannan may be partlysubstituted by various hydrophobic groups such as, but not limited to,long alkyl chains and cholesterol.

The nanoparticles or microparticles of the present invention maycomprise modified starch molecules with grafted fatty acid moieties. Thefatty acid may be grafted on to starch using potassium persulfate, forexample, as a catalyst. In another embodiment, the invention alsoencompasses surface-modification of nanoscale starch particles using,for example, stearic acid chloride (a hydrophobe), poly(ethylene glycol)or methyl ether (a hydrophilic molecule). In another embodiment, themodified starch may be an acryloyl-modified starch or anacryloyl-modified hydroxyethyl starch.

In an advantageous embodiment of the invention, the polysaccharide wasfirst derivatized to introduce aldehydic or carboxylic groups on theside chain. These groups were then crosslinked to produce more stablethree-dimensional networks.

In an advantageous embodiment, the particles are crosslinked to formhydrogels. Crosslinking may be performed using free radical initiatorssuch as persulfate salts, or redox systems involving ascorbic acid, or anaturally occurring crosslinker such as genipin. Ionic crosslinking isalso explored. Anionic polysaccharides such as gellan can be used forionic crosslinking, instead of chemicals such as borax which may not bedesirable in a food formulation.

The present invention further relates to the preparation of hydrogels.In an advantageous embodiment, a blend of hydrophobically modifiedpolysaccharide such as, but not limited to, hydroxypropyl cellulose anda carboxy containing polysaccharide such as, but not limited to,alginate or carboxymethyl cellulose may be used to prepare the hydrogelparticles of the present invention. The hydrophobically modifiedpolysaccharide results in spontaneous particle formation due to phaseseparation in water, while the polysaccharide containing carboxylic acidgroups imparts a pH-responsive behavior and will also increaseintestinal transit time. In one embodiment, nanoparticle suspensions maybe synthesized by self-assembly of chitosan and carboxymethyl cellulosehydrolysates. The polymers are hydrolyzed with the enzymes chitosanaseand cellulase, respectively. Electrostatic interactions between thecarboxylate groups of carboxymethyl cellulose with the amino groups ofchitosan result in spontaneous formation of nanoparticles by mixingsolutions of the two polymers. Particle size depended on the mixingratio of the solutions, and also by the molecular weight of thepolymers. It was necessary to hydrolyze the polymers and lower themolecular weight before mixing in order to prevent the formation ofmacroscopic gel.

In another embodiment, hydrogels may be prepared from mixtures of acidicpolysaccharides such as, but not limited to, alginates, and basicpolysaccharides such as, but not limited to, oligosaccharide derivativesof chitosan; a basic polysaccharide such as, but not limited to,chitosan and anionic polysaccharide such as, but not limited to,hyaluronic acid; alginate and oxidized alginate blended with chitosan;grafted agar and sodium alginate blend with acrylamide; gellanco-crosslinked with scleroglucan; photocrosslinked modified dextran;starch reacted with glycidyl methacrylate; or polymerizable saccharidemonomers, such as sucrose, created by reaction of the sugar with epoxyacrylate, or methacryloyl chloride and acetyl chloride.

Crosslinking of polysaccharides containing hydroxyl groups, e.g. starch,hydroxyalkyl starch, hydroxyalkyl cellulose, etc., can be achieved usinga variety of reagents including bis-epoxides, divinyl sulfone,N,N′-carbonyldiimidazole, cyanuric chloride, terephthaloyl chloride,carbon disulfide, formaldehyde, and glutaraldehyde as shown in FIGS. 8Aand 8B [Park, H.; Park, K.; Shalaby, W. S. W. Biodegradable Hydrogelsfor Drug Delivery, Technomic Publishing Company: Lancaster, Pa., 1993].Crosslinking to form macroscopic hydrogels may be readily achieved usingthese reagents. Kabra et al. [Kabra, B. G.; Gehrke, S. H.; Spontak, R.J. Microporous, responsive hydroxypropyl cellulose gels. 1. Synthesisand microstructure. Macromolecules 1998, 31, 2166-2173] have useddivinyl sulfone crosslinker to prepare macrogels of hydroxypropylcellulose. The synthesis of hydrogel nano- or microparticles, on theother hand, requires careful selection of reaction conditions to preventprecipitation (due to colloidal instability) or macrogel formation. Thedifference between a macrogel and a colloidally-stable microgelsuspension is evident from the photographs of two crosslinked samples ofhydroxypropyl cellulose polymer, shown in FIG. 9. Both the vials shownin FIG. 9 contain about 4% (w/v) of hydroxypropyl cellulose in water.The vial labeled ‘1’ shows a macrogel, while the vial labeled ‘2’contains a colloidally stable microgel suspension of hydroxypropylcellulose To prevent macrogel formation and colloidal aggregation, thepolysaccharide concentration has been kept fairly low (below about 1 wt%) in the crosslinking reactions. Cai et al. [Cai, T.; Hu, Z.; Marquez,M. Synthesis and self-assembly of nearly monodisperse nanoparticles of anaturally occurring polymer. Langmuir 2004, 20, 7355-7359] have preparednanoparticles of crosslinked hydroxypropyl cellulose using divinylsulfone crosslinker at 0.05 wt % polymer concentration. The toxicity ofdivinyl sulfone is of concern in synthesizing formulations forcontrolled release of nutrients. FIGS. 8A and 8B depict crosslinking ofpolysaccharides containing hydroxyl groups. P represents apolysaccharide segment.

The transport of small molecules such as glucose through polysaccharidehydrogels has been investigated for cell encapsulation and tissueengineering [McEntee, M.-K. E.; Bhatia, S. K.; Tao, L.; Roberts, S. C.;Bhatia, S. R. Tunable transport of glucose through ionically-crosslinkedalginate gels: effect of alginate and calcium concentration. J. Appl.Polym. Sci. 2008, 107, 2956-2962]. Ionically-crosslinked alginatehydrogel beads, with an average bead diameter of 2 mm, were preparedusing alginate and calcium chlorides. The researchers found a two-steprelease profile for glucose over a time range of 20-50 min. It should benoted that the release rates were measured by suspending theglucose-loaded spheres in pure water. The large difference in theconcentration of glucose inside the sphere and the suspending fluid(pure water) resulted in a relatively rapid release of sugar (withinabout 50 min after suspension).

Covalent-crosslinking is expected to impart greater stability (againstpremature disintegration) to the hydrogel spheres, in the wide range ofpH and ionic strength conditions that are encountered in the GI tract,than ionically-crosslinked hydrogels. When trisodium metaphosphate isused as the crosslinking agent, covalent-crosslinks are formed. Therelease rate of nutrients is tuned by controlling the crosslink densityof the microspheres. More importantly, the release rate depends on theconcentration of the nutrients outside the particles, in the aqueousphase of the suspension. Applicants' dispersions contain a relativelyhigh sugar concentration in the aqueous phase. Diffusion of nutrientsfrom the hydrogel microparticles occurs only when the nutrients getdepleted from the aqueous phase. Hence, the particles act as reservoirsof sugar and supply nutrients within the intestinal lumen over a timeperiod significantly beyond the duration reported in the study usingionically-crosslinked alginate beads (˜50 min) [McEntee, M.-K. E.;Bhatia, S. K.; Tao, L.; Roberts, S. C.; Bhatia, S. R. Tunable transportof glucose through ionically-crosslinked alginate gels: effect ofalginate and calcium concentration. J. Appl. Polym. Sci. 2008, 107,2956-2962]. In Applicants' formulations, the nutrients dissolved in theaqueous phase will be initially absorbed across the intestinalephithelium. The microparticles release entrapped nutrients at low ratesinitially (because of low concentration gradient), and at a faster ratewhen the aqueous phase nutrients are depleted (because of a greaterconcentration difference).

Acceptable molecular weights for polymers used in the present inventionmay be determined by a person of ordinary skill in the art accountingfor factors such as the desired polymer degradation rate, physicalproperties such as mechanical strength and rate of dissolution ofpolymer in solvent. Typically, an acceptable range of molecular weightsis of about 2,000 Daltons to about 2,000,000 Daltons, about 3,000Daltons to about 1,900,000 Daltons, about 4,000 Daltons to about1,800,000 Daltons, about 5,000 Daltons to about 1,700,000 Daltons, about6,000 Daltons to about 1,600,000 Daltons, about 7,000 Daltons to about1,500,000 Daltons, about 8,000 Daltons to about 1,400,000 Daltons, about9,000 Daltons to about 1,300,000 Daltons, about 10,000 Daltons to about1,200,000 Daltons, about 12,000 Daltons to about 1,100,000 Daltons,about 13,000 Daltons to about 1,000,000 Daltons, about 14,000 Daltons toabout 900,000 Daltons, about 15,000 Daltons to about 800,000 Daltons,about 16,000 Daltons to about 700,000 Daltons, about 17,000 Daltons toabout 600,000 Daltons, about 18,000 Daltons to about 500,000 Daltons,about 19,000 Daltons to about 400,000 Daltons, about 20,000 Daltons toabout 300,000 Daltons, about 21,000 Daltons to about 200,000 Daltons,about 22,000 Daltons to about 100,000 Daltons, or about 23,000 Daltonsto about 50,000 Daltons. In one embodiment, the polymer is abiodegradable polymer or copolymer.

In another preferred embodiment, the nutritional supplements can beencapsulated in microparticles or microspheres. These particlesoptionally comprise surfactants such as a cationic or anionic surfactantthat is entrapped and fixed to the particle surface. The bioadhesiveproperties of the microparticles are attributed to the chargedsurfactants entrapped on the particle surface as the hydrophobic ends ofthe surfactants are embedded in the solid core and the hydrophilic endsare exposed on the surface of the microparticles.

Bioadhesive substances, also denoted mucoadhesive substances, aregenerally known to be materials that are capable of being bound to abiological membrane and retained on that membrane for an extended periodof time. Compared with conventional controlled release systems,bioadhesive controlled release systems have the following advantages: i)a bioadhesive controlled release system localizes a biological activeingredient in a particular region, thereby improving and enhancing thebioavailability for active ingredients which may have poorbioavailability by themselves, ii) a bioadhesive controlled releasesystem leads to a relatively strong interaction between a bioadhesivesubstance and a mucosa, such an interaction contributes to an increasingcontact time between the controlled release system and the tissue inquestion and permits localization of the active released from thecontrolled release system to a specific site, iii) a bioadhesivecontrolled release system prolongs delivery of biological activeingredients in almost any non-parenteral route, iv) a bioadhesivecontrolled release system can be localized on a specific site with thepurpose of local therapy, v) a bioadhesive controlled release system canbe targeted to specific diseased tissues, and vi) a bioadhesivecontrolled release system is useful when conventional approaches areunsuitable, such as for certain biological active ingredients which arenot adequately absorbed.

The microparticles can also include at least one co-surfactant. Theco-surfactant can be a natural biologically compatible surfactant or apharmaceutically acceptable non-natural surfactant. The co-surfactantassists in maintaining particles within the desired size range andpreventing their aggregation. The co-surfactant comprises less thanabout 5%, less than about 4%, less than about 3%, less than about 2%,preferably less than about 1%, less than about 0.9%, less than about0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%,less than about 0.4%, less than about 0.3%, less than about 0.2% andmore preferably less than about 0.1% by weight of the particle.

The microparticles are preferably formed as an aqueous continuous phasesuspending a colloidal phase of submicron particles. The aqueouscontinuous phase of the particle suspension can contain antioxidants,preservatives, microbicides, buffers, osmoticants, cryoprotectants, andother known pharmaceutically useful additives or solutes.

The microparticles sustain the release rate of nutritional supplementsfor an extended period of time. For example, the micro particles sustainthe release of nutritional supplements for a period between about 1minute and twelve hours.

The use of microparticles which provide varying rates of nutrientrelease are contemplated. For example, the kinetics of nutrient-releasemay be any of the following: (i) a steady-state or zero-order releaserate in which there is a substantially uniform rate of releasethroughout; (ii) a first-order release rate in which the rate of releasedeclines towards zero with time; and (iii) a delayed release in whichthe initial rate is slow, but then increases with time.

The term “bioadhesion” relates to the attachment of a material to abiological substrate such as a biological membrane. The term“mucoadhesive substance” is in accordance with the generally acceptedterminology and is used synonymously with the term “a bioadhesivesubstance”.

A cationic surfactant is incorporated on an outer surface of themicroparticle to form a bioadhesive microparticle. The surfactant isentrapped and fixed to the particle surface and forms a coating at theinterface surrounding the particle core. The interface surrounding thecore is hydrophobic. The cationic surfactant also stabilizes the outersurface of the hydrophobic core component of the microparticles, therebypromoting a more uniform particle size.

Examples of surface active materials that are capable of strong bondingto the negatively charged and hydrophilic surfaces of tissues arepreferable for use as cationic charged surfactants. Suitable surfaceactive materials include straight-chain alkylammonium compounds, cyclicalkylammonium compounds, petroleum derived cationics, and polymericcationic materials. Cetylpyridinium chloride was found to exhibit strongbioadhesive properties on biological surfaces, and is a preferredsurface active material. The surfactant is present in a proportion ofabout 0.01% to about 5%, preferably about 0.05% to about 2%, by weightof the suspension.

Straight-chain alkylammonium compounds are cationic surface activematerials in which one or more hydrophobic alkyl groups are linked to acationic nitrogen atom. The linkage can also be more complex as, forexample, in R—C(═O)—NHCH₂CH₂CH₂N(CH₃)₂. Alternatively, the cationicsurface active material can contain more than one cationic nitrogen atomsuch as the class of compounds of R—NHCH₂CH₂CH₂NH₂ and derivativesthereof. Representative examples of suitable compounds for the cationicsurfactant include, but are not limited to: cetyl trimethylammoniumchloride (CTAB), hexadecyltrimethylammonium bromide (HDTAB), stearyldimethylbenzylammonium chloride, lauryl dimethylbenzylammonium chloride,cetyl dimethylethylammonium halide, cetyl dimethylbenzylammonium halide,cetyl trimethylammonium halide, dodecyl ethyldimethylammonium halide,lauryl trimethylammonium halide, coconut alkyltrimethylammonium halide,and C8-C20 N,N-dialkyldimethylammonium halide.

Other suitable compounds for the cationic surfactant include, but arenot limited to, bis(hydrogenated tallow alkyl)dimethylammonium chloridewhich is known to adsorb onto the surface with hydrophobic groupsoriented away from it, 2-hydroxydodecyl-2-hydroxyethyl dimethyl ammoniumchloride [CAS no. xx] andN-octadecyl-N,N′,N′-tris-(2-hydroxyethyl)-1,3-diaminopropanedihydrofluoride [CAS no. 6818-37-7].

Surface-active quaternary ammonium compounds in which the nitrogen atomcarrying the cationic charge is part of a heterocyclic ring can be usedas the cationic surfactant. Examples of suitable compounds arelaurylpyridinium chloride, bromide laurylpyridinium,tetradecylpyridinium bromide, and cetylpyridinium halide where thehalide is selected from chloride, bromide or fluoride.

Polymeric amines which can be used as the cationic surfactant comprise aclass of polymers containing ionic groups along the backbone chain andexhibit properties of both electrolytes and polymers. These materialscontain nitrogen, of primary, secondary, tertiary or quaternaryfunctionality in their backbone and may have weight average molecularweights as low as about 100 or higher than about 100,000. Suitablepolymeric amines useful as a cationic surfactant include, but are notlimited to, polydimeryl polyamine available from General Mills ChemicalCo., polyamide, polyacrylamides, polydiallyldimethylammonium chloride,polyhexamethylene biguanide compounds, and also other biguanides, forexample those disclosed in U.S. Pat. Nos. 2,684,924, 2,990,425,3,183,230, 3,468,898, 4,022,834, 4,053,636 and 4,198,425, hereinincorporated by reference into this application,1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, such as“Polybrene” manufactured by Aldrich, polyvinylpyrrolidone and theirderivatives, polypeptides, poly(allylamine) hydrochloride,polyoxyethylenated amines, and polyethyleneimine, such as “Polymin”manufactured by BASF.

Suitable polymeric materials for the cationic surfactant also includesurface active cationic polymers prepared by converting a fraction ofthe amino groups to their acyl derivatives. For example, thepolyethyleneimine is first condensed with less than the stoichiometricquantity of acid halides thus alkylating some of the amino groups andthe remaining amino groups are then condensed with hydrogen halides suchas hydrogen chloride or, preferably, hydrogen fluoride. The surfaceactivity of these compounds varies with the number of amino groups whichare acylated and with the chain length of the acylating group RCO. Thecondensation reaction can be performed with stearic or oleic acidchlorides in the presence of a solvent containing metal fluoride,preferably silver fluoride, in such a manner that metal chloride formedin the reaction precipitates from the solvent.

Also suitable, for the purpose of this invention, are cationicderivatives of polysaccharides such as dextran, starch or cellulose, forexample, diethylaminoethyl cellulose. Examples of applicable copolymersbased on acrylamide and a cationic monomer are available from HerculesInc. under the trade name RETEN including RETEN 220, or from NationalAdhesives under the trade name FLOC AID including FLOC AID 305. Otheruseful acrylamide-based polyelectrolytes are available from AlliedColloids under the trade name PERCOL. Further examples of suitablematerials are cationic guar derivatives such as those sold under thetrade name JAGUAR by Celanese-Hall.

In another preferred embodiment, the microparticles comprise ahydrophobic core which is preferably formed of a biodegradablehydrophobic materials having barrier properties. Suitable, nontoxic,pharmaceutical solid core materials are inert hydrophobic biocompatiblematerials with a melting range between about 50° C. and about 120° C.,between about 60° C. and about 110° C., between about 70° C. and about100° C. or between about 80° C. and about 90° C. Examples include, butare not limited to, natural, regenerated, or synthetic waxes including:animal waxes, such as beeswax; lanolin and shellac wax; vegetable waxessuch as carnauba, candelilla, sugar cane, rice bran, and bayberry wax;mineral waxes such as petroleum waxes including paraffin andmicrocrystalline wax; cholesterol; fatty acid esters such as ethylstearate, isopropyl myristate, and isopropyl palmitate; high molecularweight fatty alcohols such as cetostearyl alcohol, cetyl alcohol,stearyl alcohol, and oleyl alcohol; solid hydrogenated castor andvegetable oils; hard paraffins; hard fats; biodegradable polymers suchas polycaprolactone, polyamides, polyanhydrides, polycarbonates,polyorthoesters, polylactic acids, and copolymers of lactic acid andglycolic acid; cellulose derivatives and mixtures thereof. Otherhydrophobic compounds which may be used in the present invention includetriglycerides, preferably of food grade purity or better, which may beproduced by synthesis or by isolation from natural sources. Naturalsources may include animal fat or vegetable oil, such as, soy oil, asource of long chain triglycerides (LCT). Other suitable triglyceridesare composed predominantly of medium length fatty acids (C10-C18),denoted medium chain triglycerides (MCT). The fatty acid moieties ofsuch triglycerides can be unsaturated, monounsaturated orpolyunsaturated. Mixtures of triglycerides having various fatty acidmoieties are also useful for the present invention. The core cancomprise a single hydrophobic compound or a mixture of hydrophobiccompounds. Hydrophobic materials are known to those skilled in the artand are commercially available, as described in the list of suitablecarrier materials in Martindale, The Extra Pharmacopoeia, 28^(th) ed.;The Pharmaceutical Press: London, 1982; pp 1063-1072. Considerations inthe selection of the core material include good barrier properties tothe active ingredients and sensory markers, low toxicity and irritancy,biocompatibility, stability, and high loading capacity for the activeingredients of interest.

An amphiphilic or nonionic co-surfactant can be used in themicroparticles of the present invention to provide improved stability.Co-surfactants can be formed of natural compounds or normaturalcompounds. Examples of natural compounds are phospholipids and cholates.Examples of normatural compounds include: polysorbates, which are fattyacid esters of polyethoxylated sorbitol sold by Unigema surfactants asTween; polyethylene glycol esters of fatty acids from sources such ascastor oil; polyethoxylated fatty acid, such as stearic acid;polyethoxylated isooctylphenol/formaldehyde polymer; poloxamers, suchas, poly(oxyethylene)poly(oxypropylene) block copolymers available fromBASF as Pluronic; polyoxyethylene fatty alcohol ethers available fromICI surfactants as Brij; polyoxyethylene nonylphenyl ethers sold byUnion Carbide as Triton N; polyoxyethylene isooctylphenyl ethers sold byUnion Carbide as Triton X; and SDS. Mixtures of surfactant molecules,including mixtures of surfactants of different chemical types, can beused in the present invention. Surfactants preferably are suitable forpharmaceutical administration and compatible with the drug to bedelivered.

Particularly suitable surfactants include phospholipids, which arehighly biocompatible. Especially preferred phospholipids arephosphatidylcholines (lecithins), such as soy or egg lecithin. Othersuitable phospholipids include phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, phosphatidic acid,cardiolipin, and phosphatidylethanolamine. The phospholipids may beisolated from natural sources or prepared by synthesis. Phospholipidsurfactants are believed to usually form a single monolayer coating ofthe hydrophobic core. The co-surfactant can be present in an amount lessthan about 5%, preferably less than about 1%, and more preferably lessthan about 0.1%, relative to the weight of hydrophobic core component.In some embodiments, one or more co-surfactants can be used.

In another preferred embodiment, the nutritional supplements comprisecompounds which modulate uptake of carbohydrates. In thegastrointestinal tract, chromium and vanadium (either individually, orpreferably in concert) modulate sugar transport (e.g., glucosetransport) by typically slowing glucose absorption. Slower glucoseabsorption slows insulin release and reduces excessive insulin responsesin response to rising blood glucose levels after a meal. This benefitspancreatic secretion of insulin by reducing both the glucose load andrate of glucose load over the initial phases of glucose detection,absorption and metabolism by the body. Reduced rates of glucose loadingreduce the stress on beta cells normally associated with the insulinresponse to rising glucose. Moreover, slower or modulated glucoseabsorption permits more time for insulin to stimulate normal sugarmetabolic routes either before glucose loading is complete, or during aslower rate of glucose loading. Consequently, insulin dependentmechanisms have more time to prepare for the arrival of sugars from theintestine. This modulation of glucose absorption improves short-terminsulin modulation in the liver, muscle, and adipose tissue. Theseeffects in the gastrointestinal tract are, in all likelihood, short-termresponses, and they are not necessarily associated with the longer-termsystemic effects of chromium and vanadium administration.

In addition, chromium and vanadium may potentially slow glucosemetabolism by interacting with the intestine, particularly theepithelium of the intestine responsible for sugar metabolism (includingabsorption). One primary mechanism for sugar transport in the gut issodium facilitated sugar transport. Such transporters are located in thelumenal membrane of the epithelium. The basolateral membrane may alsohave an additional sugar transporter that facilitates transport out thecell and into the blood. For net sugar absorption from the lumen of thegut to the blood, sodium facilitated sugar transport generally requiresa sodium concentration favorable to the diffusion of sodium into theepithelium cell from the lumen. This concentration gradient is largelygenerated by the active transport of the Na/K ATPase in the epitheliumcells, which generally transports three sodium atoms out of the cell tothe blood side of the epithelium in exchange for two sodium atoms in thereverse direction.

Each cycle of the pump requires hydrolysis of one ATP to transportsodium and potassium against their respective concentration gradients.The hydrolysis reaction requires a divalent cation, typically magnesium.In many instances, however other divalent cations may substitute orenter into the hydrolysis reaction with varying degrees of catalyticactivity or inhibition. Substitution of trivalent cations for divalentcations in the cycle generally leads to significant inhibition of thepumping activity and/or dephosphorylation from the phosphoenzymeintermediate state. Chromium may thus inhibit the Na/K ATPase activityby substituting for magnesium and thereby inhibiting, relative tomagnesium, catalytic and transport activity, giving rise to a decreasedsodium gradient across the lumenal membrane. The reduced gradienteffects sugar transport by reducing the thermodynamic and kinetic forcesfavoring sugar entry from the gut.

In addition, during the hydrolysis of ATP in the catalytic cycle of theNa/K ATPase, a phosphoenzyme intermediate (EP) is formed betweenphosphate and an aspartic acid at the active site of APTase. Thiscovalent EP is transient and is chemically distinct from phosphorylatedproteins associated with kinases and phosphatases, which have also beenshown to be affected by vanadium. Formation of EP in the catalytic cyclefor Na/K ATPase is inhibited by vanadate present at low concentrationsof less than 1 micromolar. Vanadate binds to the active site as atransition state analog of phosphate in a vanadyl-enzyme, or EV complex,rather than EP. The EV complex is highly stable, as the kinetics of lossof vanadate from the EV complex is relatively slow. Vanadate may thuseffectively inhibit the Na/K ATPase by disrupting catalysis, through theformation of EV, giving rise to a decreased sodium gradient across thelumenal membrane. Consequently, the reduced gradient reduces sugar entryfrom the intestine.

Chromium and vanadium also operate at the systemic level afterabsorption of the two transition metals from the gut. Major sites ofactivity include the liver, muscle and adipose tissue. Vanadium may haveparticular activity with respect to phosphorylation systems, includingthe many phosphorylated proteins responsible for modulating metabolism.Chromium may also modulate metabolism at the cellular level. Thesesystemic effects generally improve the action of insulin and/ormetabolic pathways associated with sugar and/or lipid metabolism.

In regard to absorption and metabolism of the subject compositions, andthe different components thereof, features of the alimentary tract mayaffect how compositions of the present invention, and methods of usingthe same, are utilized when ingested orally. The elements of thealimentary tract, including the gastrointestinal tract, may affect thedosage required for any such modality. Such features are well known tothose of skill in the art.

In another preferred embodiment, the nutritional compositions areformulated into unit dosage forms such as tablets, caplets, powder,granules, beads, chewable lozenges, capsules, liquids, aqueoussuspensions or solutions or similar dosage forms, using conventionalequipment and techniques known in the art. Such formulations typicallyinclude a solid, semisolid, or liquid carrier. Exemplar carriers includelactose, dextrose, sucrose, sorbitol, mannitol, sutarches, gum acacia,calcium phosphate, mineral oil, cocoa butter, oil of theobroma,alginates, tragacanth, gelatin syrup, methyl cellulose, polyoxyethylenesorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate,talc, magnesium stearate, and the like.

Other formulations suitable for oral administration may be in the formof capsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), each containing a predetermined amount of asupplement or components thereof as an active ingredient. A supplementor components thereof may also be administered as a bolus, electuary, orpaste.

In other formulations, the nutritional supplements are provided inbeverages. The beverages of this invention can be carbonated beveragese.g., flavored seltzer waters, soft drinks or mineral drinks, as well asnon-carbonated juices, punches and concentrated forms of thesebeverages. Beverages, especially juice and cola beverages, which arecarbonated in the manner of soft drinks, as well as “still” beveragesand nectars and full-strength beverages or beverage concentrates whichcontain at least about 45% by weight of juice are also contemplated.

By way of example, the fruit juices and fruit flavors used here ininclude grape, pear, passion fruit, pineapple, banana or banana puree,apricot, orange, lemon, grapefruit, apple, cranberry, tomato, mango,papaya, lime, tangerine, cherry, raspberry, carrot and mixtures thereof.Additionally, artificial flavors, e.g. cola, or natural flavors derivedfrom these juices can be used in the beverages. Chocolate flavors andother non-fruit flavors can also be used to make beverages containingthe vitamin and mineral supplement. Additionally, milk, obtained fromcows or synthetic, is a contemplated beverage to which the powdercompositions of this invention can be added. The milk may itself includeother beverage components, in particular flavors such as chocolate,coffee, or strawberry. As used herein, the term “juice product” refersto both fruit and vegetable juice beverages and fruit and vegetablejuice concentrates which comprise at least about 45% fruit juice.Vegetable when used herein includes both nonfruit edible plant partssuch as tubers, leaves, rinds, and also if not otherwise indicated, anygrains, nuts, beans, and sprouts which are provided as juices orbeverage flavorings.

In one preferred embodiment, sport beverages can be supplemented by thepowder compositions of the present invention. Typical sport beveragescontain water, sucrose syrup, glucose-fructose syrup, and natural orartificial flavors. These beverages can also contain citric acid, sodiumcitrate, monopotassium phosphate, as well as other materials which areuseful in replenishing electrolytes lost during perspiration.

Tables 2 and 3 depict components of representative sports and energydrink products.

TABLE 2 Representative energy drink products. The ‘x’ indicates that anunspecified amount of the ingredient is present in the formulation.Monster Energy Red Bull RockStar Container Size 16 oz 8.3 oz 8, 16, or24 oz Serving size 8 oz 8.3 oz 8 oz Vitamin B2 1.7 mg 3.4 mg Vitamin B320 mg 100 mg 20 mg Vitamin B5 10 mg Vitamin B6 2 mg 125 mg 2 mg VitaminB12 6 μg 240 μg 6 μg Sodium 180 mg 200 mg 40 mg Potassium Folic acidMagnesium Zinc Taurine 1000 mg 1000 mg Penax Ginseng extract 200 mg 25mg Energy Blend 2500 mg 1350 mg L-Carnitine x 25 mg Glucose x Caffiene xx 80 mg Guarana x 25 mg Inositol x 25 mg Glucuronolactone x Maltodextrinx Ginko biloba extr 150 mg Milk thistle 20 mg Muira pauma extractCatuaba extract Epimedium extract Yerba mate extract Ingredients WaterCarbonated water x x x Sucrose x x x Fructose Fructose-glucose syrupGlucose x x x Acelsulfame x Aspartame/Sucralose x x TrehaloseWhey-protein isolate Citric acid x x Phoshoric acid Lactic acidMagnesium carbonate Vitamin E acetate Salt x Monopotassium phosphateEster gum Sodium ascorbate Sodium citrate x x x Flavor x x x Benzoicacid x x Sorbic acid x x Ascorbic acid x L-Carnitine x x Niacinamide x xx Calcium pantothenate x x Pantothenic acid Calcium disodium Pyroxidinehydrochloride x x Magnesium lactate Calcium lactate Gum acaciaCyanocobalamin x x Potassium citrate Modified food starch Hibiscusextract Calcium phosphate Gum Arabic Vanocobalamin Red clover flowerextract Magnesium oxide Zinc picolinate Muira pauma extr Epimediumsagittatum extract Guarana seed extract x Yerba mate extract Catuabaextract Folic acid Taurine x x Penax Ginseng extract x Glucuronolactonex x Inositol x x Riboflavin x Maltodextrin x

TABLE 3 Representative sports drink products Life Propel Vitamin WaterFunction: Fit Water (Goji Alternative Gatorade Accelerade Water ReviveMelon) Energy Container Size  20 oz  20 oz 18.9 oz 20 oz 20 oz 16.9 ozServing size  8 oz  8 oz 8 oz 8 oz  8 oz 8 oz Vitamin B2 Vitamin B3 20mg 10 mg Vitamin B5 20 mg Vitamin B6 12.5 mg 12.5 mg  5 mg 1 mg VitaminB12 75 μg 60 μg 30 μg 3 μg Sodium 110 mg 120 mg 35 mg 20 mg 120 mgPotassium  30 mg  15 mg 140 mg Folic acid 200 μg Magnesium 20 mg Zinc4.95 mg Taurine Penax Ginseng extract Energy Blend 300 mg  L-Carnitine Glucose  Caffiene x  Guarana x  Inositol  Glucuronolactone Maltodextrin  Ginko biloba extract  Milk thistle  Muira pauma extract x Catuaba extract x  Epimedium extract x  Yerba mate extract xIngredients Water x x x x x x Carbonated water Sucrose x x x x x xFructose x Fructose-glucose syrup x Glucose Acelsulfame xAspartame/sucralose x Trehalose x Whey-protein isolate x Citric acid x xx x x x Phoshoric acid x Lactic acid x Magnesium carbonate x Vitamin Eacetate x 10% x RDA Salt x x x Ester gum x x x Sodium ascorbate x Sodiumcitrate x x x Flavor x x x x x x Benzoic acid Sorbic acid Ascorbic acidx x x L-Carnitine Niacinamide x x x x Calcium pantothenate x xPantothenic acid x Calcium disodium x Pyroxidine hydrochloride x x x xMagnesium lactate Calcium lactate x Gum acacia x Cyanocobalamin x xPotassium citrate x Modified food starch x Hibiscus extract x Calciumphosphate x Gum Arabic x Vanocobalamin x Red clover flower extract xMagnesium oxide x Zinc picolinate x Muira pauma extract x Guarana seedextract x Yerba mate extract x Catuaba extract x Folic acid x TaurinePenax Ginseng extr. Glucuronolactone Inositol Riboflavin Maltodextrin

As used herein, the term “juice beverage” refers to a fruit or vegetablejuice product which is in a single-strength, ready-to-serve, drinkableform. Juice beverages of the present invention can be of the“full-strength” type which typically comprise at least about 95% juice.Full strength juice beverages also include those products of 100% juicesuch as, for example, orange, lemon, apple, raspberry, cherry, apricot,pear, grapefruit, grape, lime, tangerine, carrot, pineapple, melon,mango, papaya, passion fruit, banana and banana puree, cranberry,tomato, carrot, cabbage, celery, cucumber, spinach, and various mixturesthereof. Juice beverages also include extended juice products which arereferred to as “nectars”. These extended juice products typicallycomprise from about 50% to about 90%, about 55% to about 85%, about 60%to about 80%, about 65% to about 75% juice, preferably, from about 50%to about 70% juice. Nectars usually have added sugars or artificialsweeteners or carbohydrate substitutes. As used herein, the term “citrusjuice” refers to fruit juices selected from orange juice, lemon juice,lime juice, grapefruit juice, tangerine juice and mixtures thereof.

As used herein, the term “juice materials” refers to concentrated fruitor vegetable juice, plus other juice materials such as juice aroma andflavor volatiles, peel oils, and pulp or pomace. As used herein, theterm “juice concentrate” refers to a fruit or vegetable juice productwhich, when diluted with the appropriate amount of water, formsdrinkable juice beverages. Juice concentrates within the scope of thepresent invention are typically formulated to provide drinkablebeverages when diluted with 3 to 5 parts by weight water.

As used herein the term “beverage concentrate” or “bottling syrup”refers to a mixture of flavors, water and from about 10% to about 60%,about 20% to about 50% or about 30% to about 40% sugar or carbohydratesubstitute, i.e. sucrose, dextrose, corn syrup solids, fructose,dextrins, polydextrose and mixtures thereof.

The flavor component of the beverages and beverage concentrates containsflavors selected from fruit flavors, vegetable flavors, botanicalflavors and mixtures thereof. As used herein, the term “fruit flavor”refers to those flavors derived from the edible reproductive part of aseed plant, especially one having a sweet pulp associated with the seed,and “vegetable flavor” refers to flavors derived from other edible partsof seed and other plants. Also included within the term “fruit flavor”and “vegetable flavor” are synthetically prepared flavors made tosimulate fruit or vegetable flavors derived from natural sources.Particularly preferred fruit flavors are the citrus flavors includingorange, lemon, lime and grapefruit flavors. Besides citrus flavors, avariety of other fruit flavors can be used such as apple, grape, cherry,pineapple, mango and papaya flavors and the like. These fruit flavorscan be derived from natural sources such as juices and flavor oils, orcan be synthetically prepared. As used herein, the term “botanicalflavor” refers to flavors derived from parts of a plant other than thefruit; i.e. derived from nuts, bark, roots and leaves, and beans such ascoffee, cocoa, and vanilla. Also included within the term “botanicalflavor” are synthetically prepared flavors made to simulate botanicalflavors derived from natural sources. Examples of such flavors includecola, tea, coffee, chocolate, vanilla, almond, and the like. Botanicalflavors can be derived from natural sources such as essential oils andextracts, or can be synthetically prepared.

The flavor component can comprise a blend of various flavors, e.g. lemonand lime flavors, cola flavors and citrus flavors to form cola flavors,etc. If desired, juices such as orange, lemon, lime, apple, grape,carrot, celery, and like juices can be used in the flavor component. Theflavors in the flavor component are sometimes formed into emulsiondroplets which are then dispersed in the beverage concentrate. Becausethese droplets usually have a specific gravity less than that of waterand would therefore form a separate phase, weighting agents (which canalso act as clouding agents) are typically used to keep the emulsiondroplets dispersed in the beverage. Examples of such weighting agentsare brominated vegetable oils (BVO) and rosin esters, in particular theester gums. See Green, L. F. Developments in Soft Drinks Technology;Applied Science Publishers: London, 1978; Vol. 1, pp 87-93, for afurther description of the use of weighting and clouding agents inliquid beverages. Besides weighting agents, emulsifiers and emulsionstabilizers can be used to stabilize the emulsion droplets. Examples ofsuch emulsifiers and emulsion stabilizers include the gums, pectins,celluloses, polysorbates, sorbitan esters and propylene glycolalginates. See Green, L. F. supra at p. 92. The particular amount of theflavor component effective for imparting flavor characteristics to thebeverages and beverage concentrates (“flavor enhancing”) can depend uponthe flavor(s) selected, the flavor impression desired, and the form ofthe flavor component.

The flavor component can comprise at least 0.05% by weight of thebeverage composition, and typically from 0.1% to 2% by weight forcarbonated beverages. When juices are used as the flavor, the flavorcomponent can comprise, on a single-strength basis, up to 25% fruitjuice by weight of the beverage, preferably from 5% to 15% juice byweight for carbonated beverages.

Carbon dioxide can be introduced into the water which is mixed with thebeverage syrup or into the drinkable beverage after dilution to achievecarbonation. The carbonated beverage can be placed into a container suchas a bottle or can and then sealed. Any conventional carbonationmethodology can be used to make the carbonated beverages of thisinvention. The amount of carbon dioxide introduced into the beveragewill depend upon the particular flavor system used and the amount ofcarbonation desired. Usually, carbonated beverages of the presentinvention contain from 1.0 to 4.5 volumes of carbon dioxide. Thepreferred carbonated beverages contain from 2 to about 3.5 volumes ofcarbon dioxide.

The present invention is also particularly suited for thesupplementation of beverages and beverage concentrates, including waterand citrus juices. The beverages can contain from 3% to 100% juice orfrom about 0.05% to about 10% of an artificial or natural flavor,particularly orange juice. The concentrated orange juice, orange juicearoma and flavor volatiles, pulp and peel oils used in the method of thepresent invention can be obtained from standard orange juice. See Nagy,S.; Shaw, P. E.; Veldhuis, M. K. Citrus Science and Technology; AVIPublishing: Westport, Conn., 1977; Vol. 2, pp 177-252 for standardprocessing of oranges, grapefruit and tangerines. (See also Nelson etal. Fruit and Vegetable Juice Processing Technology, 3rd ed.; AVIPublishing: Westport, Conn., 1980; pp. 180-505, for standard processingof noncitrus juices such as apple, grape, pineapple, etc. to providesources of juice and juice materials for noncitrus juice products).

Juices from different sources are frequently blended to adjust the sugarto acid ratio of the juice. Different varieties of oranges can beblended or different juices can be blended to get the desired flavor andsugar to acid ratio. A sugar to acid ratio of from about 8:1 to about20:1 is considered acceptable for fruit juices. However, preferred sugarto acid ratios are typically from about 11:1 to about 15:1, especiallyfor citrus juices. Sweeteners include the sugars normally present injuice products, for example glucose, sucrose, and fructose. Sugars alsoinclude high fructose corn syrup, invert syrup, sugar alcohols,including sorbitol, refiners syrup, and mixtures thereof. In addition tosugar, extended juice beverages of the present invention can containother sweeteners. Other suitable sweeteners include saccharin,cyclamates, acetosulfam, L-aspartyl-L-phenylalanine lower alkyl estersweeteners (e.g. aspartame). A preferred sweetener for use in suchextended juice products is aspartame. For single-strength juicebeverages, the sugar content can range from about 2° to about 16° Brix(16° Brix means the juice contains about 16% soluble solid, and so on).Typically, the sugar content of such beverages depends upon the amountof juice contained herein.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the supplement or componentsthereof is mixed with one or more pharmaceutically-acceptable carriers,such as sodium citrate or dicalcium phosphate, and/or any of thefollowing: (1) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; (2) binders, such as, forexample, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the supplement or componentsthereof moistened with an inert liquid diluent. Tablets, and other soliddosage forms, such as dragees, capsules, pills and granules, mayoptionally be scored or prepared with coatings and shells, such asenteric coatings and other coatings well known in thepharmaceutical-formulating art.

Tablets and other solid dosage forms may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which maybe dissolved in sterile water, or sonic other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which may be used include polymeric substancesand waxes. The active ingredient may also be in micro-encapsulated form,if appropriate, with one or more of the above-described nutritionalsupplements.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the supplement or component, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions may also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions, inaddition to the supplement or components thereof, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

The composition of the invention can be administered as a capsule ortablet containing a single or divided dose of the inhibitor. Preferably,the composition is administered as a sterile solution, suspension, oremulsion, in a single or divided dose. Tablets may contain carriers suchas lactose and corn starch, and/or lubricating agents such as magnesiumstearate. Capsules may contain diluents including lactose and dried cornstarch.

A tablet may be made by compressing or molding the active ingredientoptionally with one or more accessory ingredients. Compressed tabletsmay be prepared by compressing, in a suitable machine, the activeingredient in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, surfaceactive, or dispersing agent. Molded tablets may be made by molding in asuitable machine, a mixture of the powdered active ingredient and asuitable carrier moistened with an inert liquid diluent.

When preparing dosage form incorporating the compositions of theinvention, the compounds may also be blended with conventionalexcipients such as binders, including gelatin, pregelatinized starch,and the like; lubricants, such as hydrogenated vegetable oil, sutearicacid, and the like; diluents, such as lactose, mannose, and sucrose;disintegrants, such as carbox.nmethylcellulose and sodium starchglycolate; suspending agents, such as povidone, polyvinyl alcohol, andthe like; absorbants, such as silicon dioxide; preservatives, such asmethylparaben, propylparaben, and sodium benzoate; surfactants, such assodium lauryl sulfate, polysorbate 80, and the like; colorants such asF.D. & C. dyes and lakes; flavorants; and sweeteners.

The invention will now be further described by way of the followingnon-limiting examples.

Example Delayed-Release Carbohydrate Formulations for AthleticPerformance Enhancement

The goal of the Example is to develop a nutrition fluid that deliverscarbohydrates and other nutrients to an athlete in a manner thatpromotes peak athletic performance. The proposed approach is to engineercontrolled release of digestible carbohydrates from aqueous dispersionof suitable micro or nanospheres. Important digestible carbohydratesinclude: the monosaccharides—glucose, fructose and galactose; thedissacharides—sucrose, maltose and lactose; and the polysaccharide,starch. Starch is broken down in to dextrins by salivary amylase (in themouth) and pancreatic amylase (in the small intestine). Dextrin is actedupon by the brush border enzymes in the small intestine, which alsoconvert the double sugars into simple sugars. The monosaccharides arefinally transported across the intestinal epithelium into thebloodstream. The proposed research seeks controlled release ofdigestible carbohydrates, especially the simple sugars, glucose andfructose, for sustained uptake into the blood.

A basic understanding of the physiology of the gastrointestinal (GI)tract is useful in the design of the delivery system. The retention timeof food in the stomach is up to 2 hours and depends, among otherfactors, on the calorific value of the meal (see, e.g., Hadi, N. A.;Giouvanoudi, A.; Morton, R.; Horton, P. W.; Spyrou, N. M. Variations ingastric emptying times of three stomach regions for simple and complexmeals using scintigraphy. IEEE Transactions on Nuclear Science 2002, 49,2328-2331). The controlled release system should be able to withstandthe acidic pH (1-3) of the stomach during gastric retention, withoutreleasing the sugar payload. Residence time in the small intestine,where most of the nutrient absorption occurs, is about 3 h. For nutrientdelivery over a longer time period, it is necessary to prolongintestinal retention which may be achieved by encapsulating the nutrientin a carrier with mucoadhesive properties. Hydrophilic polymerscontaining carboxylic acid groups exhibit good mucoadhesive properties.A key step in the design of a controlled release system for sugar is theselection of a carrier material for encapsulating carbohydrates.Polysaccharides and their derivatives are polymers of choice as carriersfor sustained-release drug delivery and scaffolds in tissue engineeringbecause of their non-toxic nature and excellent biocompatibility (see,e.g., Dumitriu, S.; Dumitriu, M. Hydrogels as support for drug deliverysystems. In Polysaccharides in Medicinal Applications; Dumitriu, S. Ed.;Dekker: New York, 1996; pp 705-764; Coviello, T.; Matricardi, P.;Marianecci, C.; Alhaique, F. Polysaccharide hydrogels for modifiedrelease formulations. J. Control. Rel. 2007, 119, 5-24 and Kong, H.;Mooney, D. J. Polysaccharide-based hydrogels in tissue engineering. InPolysaccharides, 2^(nd) ed.; Dumitriu, S., Ed.; Dekker: New York, 2005;pp 817-837). They have also been used for flavor encapsulation in foodformulations (see, e.g., Madene, A.; Jacquot, M.; Scher, J.; Desobry, S.Flavour encapsulation and controlled release—a review. InternationalJournal of Food Science and Technology 2006, 41, 1-21).

Blends of polysaccharides are used to synthesize aqueous dispersions ofmicro- or nanoparticles. Hydrophobically modified polysaccharides suchas hydroxypropyl cellulose or hydroxyethyl cellulose are known tospontaneously form nanoparticles in water. Interpenetrating polymernetworks of these polymers, with polysaccharides containing carboxylicacid groups, are synthesized. FIG. 1 shows the chemical structures ofthree important carboxy-containing polysaccharides. The monomeric unitof the carboxymethylcellulose backbone consists of D glucose residueslinked through β-(1→4) bonds. Alginates are composed of (1→4)-linkedβ-D-mannuronic acid and α-L-guluronic acid monomers which vary in amountand sequential distribution along the polymer chain depending on thesource of alginate. Hyaluronic acid is a straight polymer consisting ofalternating (1→4)-linked 2-acetamide-2-deoxy-β-D-glucose and (1→3)linked β-D-glucuronic acid.

To increase stability of the particles in the GI tract, the particlesare crosslinked to form hydrogels. Different crosslinking mechanisms areinvestigated to achieve the desired release kinetics. Crosslinking isperformed using free radical initiators such as persulfate salts, orredox systems involving ascorbic acid, or a naturally occurringcrosslinker such as genipin. Ionic crosslinking is also explored.Anionic polysaccharides such as gellan can be used for ioniccrosslinking, instead of chemicals such as borax which may not bedesirable in a food formulation.

It is expected that the carboxy containing hydrogel particles are in acollapsed state in the acidic environment of the stomach. Hence, theencapsulated sugar molecules are retained within the particles in thestomach. The hydrogel particles will achieve an expanded state when theyreach the small intestine (pH 5-7), and will release the encapsulatedsugar at a rate faster than that in the stomach.

Several researchers have investigated the synthesis of polysaccharideparticles and hydrogels for controlled release. Most of these studieswere, however, focused on incorporating relatively hydrophobic drugs orprotein macromolecules in the carriers. An objective of the proposedresearch is to encapsulate small hydrophilic molecules such as sugars.The equilibrium partitioning of sugar molecules between the hydrogelparticles and the aqueous phase is determined. Due to similarities inthe chemical structures of the polysaccharide carrier and theencapsulated monosaccharides, it is expected that the encapsulationefficiency of polysaccharide hydrogels are higher than those of otherhydrogels.

There are only a few studies that have reported delayed release systemsfor carbohydrates. Fox and Allen (Fox, G. J.; Darlene, A. Method andcomposition for controlling the release of carbohydrates byencapsulation. U.S. Pat. No. 5,536,156, Jul. 16, 1996) have coatedcarbohydrate microparticles with an edible delayed-release coating. Thecoated carbohydrate, when orally ingested, causes a time delayed releaseof the carbohydrate into the digestive system. The coated particles were30 to 100 μm in size and were stored in solid particulate form. Incontrast, Applicants seek to develop controlled release particles thatare dispersed in an aqueous medium. Lake and Smith (Lake, M.; Smith, U.Composition and method for long-term glycemic control. Int. Pat. Appl.WO/2006/022585, Feb. 3, 2006) have reported the preparation of starchgranules that can be used for improved long-term control of bloodglucose in a diabetic patient. The delayed-release starch formulationwas designed to reduce the incidence of nocturnal hypoglycemia, whereinthe patient would ingest a therapeutic amount of starch granules atbedtime. Zecher (Zecher, D. C. Controlled release carbohydrate embeddedin a crosslinked polysaccharide. Int. Pat. Appl. WO/2000/032064, Aug. 6,2000) has reported a similar controlled release carbohydrate compositionconsisting of covalently crosslinked polysaccharides. However, thecrosslinked carbohydrates were not in a particulate form, and were notin the form of aqueous suspensions.

The following sections will describe methods for the synthesis ofpolysaccharide hydrogels.

Hydrophobized polysaccharides are highly promising in the synthesis ofnanoparticles because of their self-assembling properties in aqueousenvironment. Akiyoshi and Sunamoto (Akiyoshi, K.; Sunamoto, J.Supramolecular assembly of hydrophobized polysaccharides. SupramolecularScience 1996, 3, 157-163) found that polysaccharides that werefunctionalized with hydrophobes such as cholesterol spontaneously formednanoparticles when dispersed in water. The size, density and colloidalstability of the nanoparticle could be controlled by tailoring thegrafting density and degree of hydrophobicity of the hydrophobe.Polysaccharides such as pullulan, dextran and mannan were partlysubstituted by various hydrophobic groups such as long alkyl chains andcholesterol. For example, pullulan with a molecular weight of 55 kDa,when functionalized with cholesterol (˜1.7 cholesterol moieties per 100units of glucose) spontaneously formed nanoparticles that were 20-30 nmin size (Akiyoshi, K.; Deguchi, S.; Tajima, H.; Nishikawa, T.; Sunamota,J. Self-assembly of hydrophobized polysaccharide: Structure of hydrogelnanoparticle and complexation with organic compounds. Proc. Japan Acad.1995, 71, 15-19). The cholesterol bearing pullulan self-aggregated toform monodisperse stable nanoparticles after ultrasonification of thesuspension in water. No coagulation occurred even after heating at 90°C. for 1 h. These nanoparticles were used for hosting hydrophobicsubstances such as antitumor adriamycin (Akiyoshi, K.; Taniguchi, I.;Fukui, H.; Sunamoto, J. Hydrogel nanoparticle formed by self-assembly ofhydrophobized polysaccharide. Stabilization of adriamycin bycomplexation. European Journal of Pharmaceutics and Biopharmaceutics1996, 42, 286-290) and various water-soluble proteins, but encapsulationof small water-soluble molecules was not reported.

Simi and Abraham (Simi, C. K.; Abraham, T. E. Hydrophobic grafted andcrosslinked starch nanoparticles for drug delivery. Bioprocess andBiosystems Engineering 2007, 30, 173-180) have grafted fatty acid on tostarch using potassium persulfate as catalyst. Nanoparticles resultingfrom the modified starch molecules were further stabilized bycrosslinking with sodium tripolyphosphate. The nanoparticles were usedfor encapsulation of a hydrophobic drug.

Thielemans et al. (Thielemans, W.; Belgacem, M. N.; Dufresne, A. Starchnanocrystals with large chain surface modifications. Langmuir 2006, 22,4804-4810) were also successful in surface-modification of nanoscalestarch particles using stearic acid chloride (a hydrophobe) andpoly(ethylene glycol) methyl ether (a hydrophilic molecule). Woo et al.(Woo, B. H.; Jiang, G.; Jo, Y. W.; DeLuca, P. P. Preparation andcharacterization of a composite PLGA and poly(acryloyl hydroxyethylstarch) microsphere system for protein delivery. Pharmaceutical Research2001, 18, 1600-1606) have reported the synthesis of polysaccharidemicrospheres using acryloyl-modified hydroxyethyl starch. Themicrospheres were investigated for their ability to load protein forcontrolled protein delivery.

Basheer et al. (Besheer, A.; Hause, G.; Kressler, J.; Mader, K.Hydrophobically modified hydroxyethyl starch: Synthesis,characterization, and aqueous self-assembly into nano-sized polymericmicelles and vesicles. Biomacromolecules 2007, 8, 359-367) reactedhydroxyethyl starch with fatty acids (lauric, palmitic, and stearicacids) under mild reaction conditions using dicyclohexyl carbodiimide(DCC) and dimethylaminopyridine (DMAP). The resulting polymersself-assembled to form 20-30 nm micelles and 250-350 nm polymericvesicles. However, chemicals such as DCC and DMAP are potentially toxicand cannot be used in edible formulations.

Chakraborty et al. (Chakraborty, S.; Sahoo, B.; Teraoka, I.; Gross, R.A. Solution properties of starch nanoparticles in water and DMSO asstudied by dynamic light scattering. Carbohydrate Polymers 2005, 60,475-481) have studied the solution properties of starch nanoparticles inwater using dynamic light scattering. The nanoparticles were obtainedfrom Ecosynthetix (Lansing, Mich.), and were synthesized from cornstarch using glyoxal as crosslinker. A mixture of starch, glycerol (18wt % of dry starch), and glyoxal (0.1-10 wt %) was extruded to obtaincrosslinked starch granules. The granules were cryogenically ground andsieved to obtain particles smaller than 150 nm in diameter. Dynamiclight scattering or the particles in water indicated two mainpopulations, with mean diameters of 40 and 300 nm, consisting ofisolated starch nanoparticles and their aggregates, respectively. Athigher concentration (˜3% w/w), a third peak appeared at around 1 μm,because of particle aggregation. Control of particle aggregation is animportant step in the design of carbohydrate nanoparticles.

A key feature of the proposed polysaccharide hydrogels is their pHresponsiveness. Ideally, the hydrogels should not swell in the acidicenvironment of the stomach, but should swell upon entry into the smallintestine and release the encapsulated sugars at a controlled rate. Thissection reviews an extreme case where the polysaccharide matrix wasinsoluble in acidic environments, while it completely dissolved athigher pH values.

Scleroglucan is a branched homopolysaccharide that gives only D-glucoseupon complete hydrolysis. The polymer consists of a main chain of(1→3)-linked β-D-glucopyranosyl units. At every third unit along themain chain, the polymer bears a single (1→6)-linked β-D-glucopyranosylunit as a branch. The glucopyranose side chain of scleroglucan wasoxidized by means of a two-step reaction: first with periodate, to forman aldehyde derivative, and then with chlorite, which resulted in thecarboxylated derivative called sclerox (see, e.g., FIG. 2 and Coviello,T.; Palleschi, A.; Grassi, M.; Matricardi, P.; Bocchinfuso, G.;Alhaique, F. Scleroglucan: A versatile polysaccharide for modified drugdelivery. Molecules 2005, 10, 6-33). By varying the ratio betweenoxidizing agent and polysaccharide, the polymer could be oxidized to adifferent extent. It was found that above a 60% oxidation, scleroxbecame sensitive to environmental conditions giving a reversible sol-geltransition mediated by pH. Permeation of model molecules occurred atdifferent rates through the sol and the gel, and consequently, releasefrom sclerox tablets showed different profiles in the two environmentssimulating the gastric and the intestinal fluids, respectively.

In acidic medium the formation of a swelled layer around the dosage formacquired a fundamental role in determining the rate of delivery, whileat higher pH values erosion and dissolution became predominant. Additionof an acid substance, such as citric acid, in the formulation reducedthe release rate in simulated intestinal fluid. The delivery rate wasstill too rapid in relation to the transit time through the GI tract.Thus another strategy was used. The polysaccharide was first derivatizedto introduce aldehydic or carboxylic groups on the side chain. Thesegroups were then crosslinked to produce more stable three-dimensionalnetworks.

Pitarresi et al. have reported crosslinking of methacrylic anhydridefunctionalized carbohydrates by UV irradiation. Hyaluronic acid wasfirst derivatized with methacrylic anhydride. Relatively lowmolecular-weight hyaluronic acid (174 kDa) was dissolved in water toform a 2% (w/v) solution. A 20-fold excess of methacrylic acid (withrespect to the moles of repeating unit of hyaluronic acid) was addedwith simultaneous addition of 5 N NaOH (to maintain the pH between 8 and9). The reaction was kept at 4° C. and stirred for 24 h. The reactionmixture was then precipitated in ethanol and the product was recoveredand purified by centrifugation and gel permeation chromatography.

Giezen et al. have disclosed a process for producing biopolymernanoparticles in which a starch or a starch derivative was crosslinkedusing dialdehyde or polyaldehyde (see, e.g., Giezen, F. E.; Jongboom, R.O. J.; Fell, J.; Gotlieb, K. F.; Boersma, A. Biopolymer nanoparticles.U.S. Pat. No. 6,677,386, Jan. 13, 2004). A plasticizer, glycerol, wasused during the process, along with an acid such as maleic acid, oxalicacid, or citric acid. It should be noted that chemicals such asdialdehydes and glycerol are not suitable as food ingredients. Thecrosslinked nanoparticles had an average particle size below 400 nm. Theviscosity of an aqueous dispersion containing 10 wt % of these particleshad a viscosity below 150 mPas (measured at a shear rate of 186 s⁻¹).

The formulation viscosity is expected to increase with an increase inparticle concentration. As a first approximation, viscosity of asuspension is related to the particle concentration through theEinstein's equation, η=η_(w)(1+2.5φ), where η is the viscosity of thedispersion, η_(w) is the viscosity of the aqueous phase, and φ is thevolume fraction of particles in the dispersion. The particle volumefraction is given by

${\varphi = \left\lbrack {1 + {\left( \frac{\rho_{p}}{\rho_{w}} \right) \cdot \left( {\frac{1}{m} - 1} \right)}} \right\rbrack^{- 1}},$

where ρ_(p) is the density of the particles, ρ_(w) is the aqueous phasedensity, and m is the mass fraction of particles in the dispersion.Dispersion viscosity also depends on the interparticle distance, H,which is the average distance between the surfaces of two neighboringparticles in the dispersion. For a population of monodisperse particleswith hexagonal close packed structure, the interparticle distance isgiven by

$H = {D\left\{ {\left( \frac{0.74}{\varphi} \right)^{1/3} - 1} \right\}}$

where D is the particle diameter. Therefore, for a given mass fractionof polymer in the dispersion (that is, a fixed φ) the dispersionviscosity is expected to be higher when the particles are smaller insize. In this Example, the viscosity of the dispersion is tailored to beclose to that of water (˜1 mPas).

Magnani et al. have synthesized polysaccharide hydrogels usingalginates, hyaluronane, and carboxymethylcellulose (see, e.g., Magnani,A.; Rappuoli, R.; Lamponi, S.; Barbucci, R. Novel polysaccharidehydrogels: characterization and properties. Polym. Adv. Technol. 2000,11, 488-495 and Barbucci, R.; Consumi, M.; Lamponi, S.; Leone, G.Polysaccharides based hydrogels for biological applications. Macromol.Symp. 2003, 204, 37-58). The crosslinking procedure consisted ofactivating the carboxylate moieties by 2-chloro-1-methylpyridiniumiodide and using 1,3-diaminopropane as a crosslinker. Aqueous solutionof the sodium hyaluronate, alginate and carboxymethyl cellulose weretransformed in carboxylic acid by treatment with a strong acid exchangeresin Dowex 50 WX8 (Fluka) at 4° C. The solution was added to a 5%tetrabutylammonium hydroxide solution to achieve a pH between 8 and 9.After lyophilization the tetrabutylammonium salt of the polysaccharidewas dissolved in dimethylformamide (DMF). A stoichiometric quantity of2-chloro-1-methylpyridinium iodide was added to the solution kept at 0°C. The crosslinking diamine was then added in excess to the solution andthe reaction mixture was maintained under stirring at room temperaturefor 4 h. The reaction was facilitated by the addition of a small amountof triethylamine, which acted as hydrogen iodide captor. Macroscopicgels were obtained. The synthesis of micro- or nanoparticles was notreported. Moreover, the chemicals involved are not suitable for foodformulation.

Kabra et al. prepared macrogels of hydroxylpropyl cellulose bycrosslinking the polymer with divinyl sulfone at a temperature above thelower critical solution temperature (LCST, 41° C.) (see, e.g., Kabra, B.G.; Gehrke, S. H.; Spontak, R. J. Microporous, responsive hydroxypropylcellulose gels. 1. Synthesis and microstructure. Macromolecules 1998,31, 2166-2173). Cai et al. used the same reaction to prepare aqueoussuspensions of hydroxylpropyl cellulose nanoparticles (see, e.g., Cai,T.; Hu, Z.; Marquez, M. Synthesis and self-assembly of nearlymonodisperse nanoparticles of a naturally occurring polymer. Langmuir2004, 20, 7355-7359). High molecular weight (˜10⁶ Da) was dissolved inan aqueous solution of sodium hydroxide (pH 12).Dodecyltrimethylammonium bormide and dinvyl sulfone were added as thesurfactant and crosslinker, respectively. The solution was heated to atemperature above the LCST for about 3 h resulting in nanoparticles withdiameters ranging from 170 nm to 430 nm. The particle concentration wasbelow 0.1 wt %. Gao et al. have also reported in situ crosslinking ofself-associated hydroxypropyl cellulose nanoparticles using divinylsulfone (see, e.g., Gao, J.; Haidar, G.; Lu, X.; Hu, Z. Self-associationof hydroxypropylcellulose in water. Macromolecules 2001, 34, 2242-2247).

De Nooy et al. have used the reaction between a carboxylic acid, analdehyde, and an isocyanide (the Passerini three-component condensation)to prepare polysaccharide hydrogels (see, e.g., De Nooy, A. E. J.;Masci, G.; Crescenzi, V. Versatile synthesis of polysaccharide hydrogelsusing the Passerini and Ugi multicomponent condensations. Macromolecules1999, 32, 1318-1320). Carboxylic acid containing carbohydrates such ascarboxymethyl cellulose or hyaluronic acid were used to prepare thehydrogels. Polysaccharides such as scleroglucan or pullulan that do notcontain carboxylic acid groups were partially oxidized using TEMPO tointroduce aldehyde and carboxylic acid groups. The Ugi condensationreaction involves an additional component, an amine. Formaldehyde orglutaraldehyde were used along with amines such as 1,5-diaminopentane orammonium chloride for the condensation reaction. Chitosan was also usedin hydrogel synthesis because of the amine groups present in itsstructure. All the gels synthesized were macroscopic gels. Micro- ornanoparticle synthesis was not reported. Moreover, aldehydes andisocyanides are generally not considered safe as food ingredients.

Covalently crosslinked hydrogels prepared using other crosslinkingreactions have also been reported. Dou et al. have synthesizedcarboxy-functionalized nanoparticles of dextran, hydropropyl cellulose,and hydroethyl cellulose (see, e.g., Dou, H.; Tang, M.; Yang, W.; Sun,K. One-pot synthesis, characterization, and drug loading ofpolysaccharide-based nanoparticles with carboxy functional groups.Colloid Polym. Sci. 2007, 285, 1043-1047). Their procedure, however,involves the use of chemicals such as nitric acid, acrylic acid, cerium(IV) ammonium nitrate, and N,N′-methylene bisacrylamide, which areunsuitable for food formulation. Yu and Hoffman have reported thesynthesis of chemically crosslinked sodium alginate/chondroitin6-sulfate hydrogels for controlled release of a model cationic protein,lysozyme (see, e.g., Yu, X. J.; Hoffman, A. S. Polysaccharide hydrogelsas drug delivery matrixes. Proceedings of the 22^(nd) InternationalSymposium on Controlled Release of Bioactive Materials, 1995; ControlledRelease Society; pp 352-353). The hydrogel was found to be in acondensed state in divalent cation solutions and in a decondensed statein PBS. Chen et al. have reviewed some polysaccharide hydrogels thatwere used for nasal delivery of peptides and proteins (see, e.g., Chen,J.; Jo, S.; Park, K. Polysaccharide hydrogels for protein drug delivery.Carbohydrate Polymers 1995, 28, 69-76). These hydrogels, which weredesigned for nasal delivery of insulin, included 45 μm starchmicrospheres (see, e.g., Illum, L.; Jorgensen, H.; Bisgaard, H.;Krogsgaard, O.; Rossing, N. Bioadhesive microspheres as a potentialnasal drug delivery system. Int. J. Pharm. 1987, 39, 189-199),epichlorohydrin crosslinked dextran (see, e.g., Edman, P.; Björk, E.Routes of delivery: Case studies: (1) Nasal delivery of peptide drugs.Adv. Drug Delivery Rev. 1992, 8, 165-177) and hyaluronic acid estermicrospheres (see, e.g., Illum, L.; Farraj, N. F.; Fisher, A. N.; Gill,I.; Miglietta, M.; Benedetti, L. M. Hyaluronic acid ester microspheresas a nasal delivery system for insulin. J. Control. Rel. 1994, 29,133-141). The microspheres of hyaluronic esters were 10 to 100 μm indiameter (see, e.g., Benedetti, L. M.; Topp, E. M.; Stella, V. J.Microspheres of hyaluronic acid esters-Fabrication methods and in vitrohydrocortisone release. J. Control. Rel. 1990, 13, 33-41).

Selection of a suitable crosslinker is a key step in the preparation ofpolysaccharide hydrogels for food formulations. Clearly, toxicity of thecrosslinking chemical precludes its use. Genipin is a naturallyoccurring crosslinker for proteins and polysaccharides, and is obtainedfrom gardenia fruit extracts. It has attracted significant interest inthe synthesis of polysaccharide hydrogels. It has low acute toxicity(LD₅₀ i.v. 382 mg/kg in mice) and is much less toxic than most otherchemical crosslinking agents such as glutaraldehyde. Its structure isshown in FIG. 2.

Meena et al. used genipin to crosslink agarose in aqueous medium at pH˜7 at ambient conditions (see, e.g., Meena, R.; Prasad, K.; Siddhanta,A. K. Preparation of genipin-fixed agarose hydrogel. J. Appl. Polym.Sci. 2007, 104, 290-296). Genipin was purchased from ChallengeBioproducts Co., Taiwan. The amount of genipin used was about 0.8 wt %of the mass of agarose. Genipin-fixed agarose showed decreased swellingin acidic medium (pH 1.2), representative of the gastric environment.The extent of swelling was 4.8 g/g, whereas the uncrosslinked agaroseswelled up to 6 g/g. The authors have reported that, under ambientconditions, maximum crosslinking was achieved after about 85 h.

Alternatively, crosslinking can be achieved using free radicals. Freeradical initiators such as ammonium persulfate are listed in GRAS listof chemicals, and can be used in food formulations.

Based on the well known borax mediated crosslinking of polymerscontaining hydroxyl groups, Palleschi et al. have synthesized hydrogelsof scleroglucan using borax (see, e.g., Palleschi, A.; Coviello, T.;Bocchinfuso, G.; Alhaique, F. Investigation of a new scleroglucan/boraxhydrogel: structure and drug release. Int. J. Pharm. 2006, 322, 13-21).They have studied the release kinetics of model drugs theophylline,vitamin B12 and myoglobin from the crosslinked hydrogels. Thesehydrogels were macroscopic gels, and not micro- or nanospheres.

Gellan can also be used as an ionic crosslinking agent. Gellan is ananionic microbial polysaccharide that is well known for its gellingproperties in the presence of counterions, especially divalent ions,like calcium. Gellan has been used as a crosslinker for scleroglucan.

Carrageenans are linear sulfated biopolymers, composed of D-galactoseand 3,6-anhydro-D-galactose units. κ-Carrageenan beads are prepared bygelling with monovalent ions (often K⁺) and sometimes divalent ions.Alginates are linear polysaccharides produced by algae, which containvarying amounts of (1→4)-linked β-D-mannuronic acid and α-L-guluronicacid residues. Mohamadnia et al. have synthesized ionically crosslinkedbeads of carbohydrate biopolymers κ-carrageenan (FIG. 5) and sodiumalginate (see, e.g., Mohamadnia, Z.; Zohuriaan-Mehr, M. J.; Kabiri, K.;Jamshidi, A.; Mobedi, H. pH-Sensitive IPN hydrogel beads ofcarrageenan-alginate for controlled drug delivery. J. Bioactive Compat.Polym. 2007, 22, 342-356 and Mohamadnia, Z.; Zohuriaan-Mehr, M. J.;Kabiri, K.; Jamshidi, A.; Mobedi, H. Ionically crosslinkedcarrageenan-alginate hydrogel beads. Journal of Biomaterials Science:Polymer Edition 2008, 19, 47-59). Alginate gelation takes place whendivalent or trivalent cations (usually Ca²⁺) interact ionically withguluronic acid residues, resulting in the formation of athree-dimensional network. Alginate-Ca²⁺ hydrogels have been studied forcontrolled release oral drug formulations (see, e.g., Bajpai, S. K.;Sharma, S. Investigation of swelling/degradation behavior of alginatebeads crosslinked with Ca²⁺ and Ba²⁺ ions. React. Func. Polym. 2004, 59,129-140).

Langer et al. have described the synthesis of interpenetrating polymernetworks consisting of ionically or covalently crosslinked hydrogels(see, e.g., Langer, R. S.; Anseth, K.; Elisseeff, J. H.; Sims, D.Semi-interpenetrating or interpenetrating polymer networks for drugdelivery and tissue engineering. U.S. Pat. No. 6,224,893, May 1, 2001).Carbohydrates such as hyaluronic acid, dextran, heparin sulfate,chondroitin sulfate, heparin, alginate, gellan and carrageenan were usedto synthesize the ionically crosslinked hydrogels. The covalentlycrosslinked hydrogels consisted of chitosan polymer and isothiocyanatecrosslinker. The hydrogels were in the form of viscous solutions capableof retaining biologically active molecules or drugs when injected invivo. The formation of micro- or nanoparticles was not reported. The useof isothiocyante crosslinker may not be appropriate for food gradehydrogel synthesis.

A blend of hydrophobically modified polysaccharide such as hydropropylcellulose and a carboxy containing polysaccharide such as alginate orcarboxymethyl cellulose is used to prepare the hydrogel particles. Thehydrophobically modified polysaccharide results in spontaneous particleformation due to phase separation in water, while the polysaccharidecontaining carboxylic acid groups imparts a pH-responsive behavior andwill also increase intestinal transit time. A review of the formation ofhydrogels (both macroscopic gels and aqueous dispersions) using a blendof polysaccharides follows.

Ichikawa et al. have synthesized nanoparticle suspensions of 0.5 wt %concentration by self-assembly of chitosan (with a degree ofdeacetylation ˜77%) and carboxymethyl cellulose hydrolysates (see, e.g.,Ichikawa, S.; Iwamoto, S.; Watanabe, J. Formation of biocompatiblenanoparticles by self-assembly of enzymatic hydrolysates of chitosan andcarboxymethyl cellulose. Biosci. Biotechnol. Biochem. 2005, 69,1637-1642). The polymers were hydrolyzed with the enzymes chitosanaseand cellulase, respectively. Electrostatic interactions between thecarboxylate groups of carboxymethyl cellulose with the amino groups ofchitosan resulted in spontaneous formation of nanoparticles just bymixing solutions of the two polymers. Particle size depended on themixing ratio of the solutions, and also by the molecular weight of thepolymers. It was necessary to hydrolyze the polymers and lower themolecular weight before mixing in order to prevent the formation ofmacroscopic gel.

Sergio et al. have reported the preparation of hydrogels from mixturesof acidic polysaccharides such as alginates, and basic polysaccharidessuch as oligosaccharide derivatives of chitosan (see, e.g., Sergio, P.;Ivan, D.; Eleonora, M. Hydrogels of polysaccharide mixtures for tissueengineering and as carriers of active compounds. Int. Pat. Appl.WO/2007/135114, Nov. 29, 2007). They have described the synthesis ofmicrocapsules with a mean diameter of 870 μm using an electrostatic‘bead generator’. An alternative chemical method of synthesizing theparticles consisted of, for example, mixing solutions of alginate andlactose derivative of chitosan prepared in 0.15 M NaCl and 10 mM HEPES(pH 7.4). The total polymer concentration was 2% and the weight ratio ofpolyanion to polycation was 3:1. The particles were typically large insize such that they could be imaged by optical microscopy.

White et al. have prepared hydrogel films (for application in the fieldsof dermatology, plastic surgery, urology and orthopaedics) using basicpolysaccharide such as chitosan and anionic polysaccharide such ashyaluronic acid (see, e.g., White, B. J.; Rodden, G. I. Compositions ofsemi-interpenetrating polymer network. Int. Pat. Appl. WO/2005/061611,Jul. 7, 2005).

Vieira et al. have prepared hydrogels of oxidized alginate and oxidizedalginate blended with chitosan, and studied the interaction of thesedrugs with the antifolate drug pyrimethamine (see, e.g., Vieira, E. F.S.; Cestari, A. R.; Airoldi, C.; Loh, W. Polysaccharide-based hydrogels:Preparation, characterization and drug interaction behavior.Biomacromolecules 2008, 9, 1195-1199). Sodium alginate was partiallyoxidized using sodium periodate to obtain 2,3-dialdehyde alginate. Theperiodate was completely removed by extensive dialysis. Gelation of2,3-dialdehyde alginate was achieved with CaCl₂ or chitosan/CaCl₂ in thepresence of borax. The synthesis of micro- or nanoparticles is notreported.

Meena et al. have discussed the synthesis of a hydrogel system based ongrafting of agar and sodium alginate blend with acrylamide (see, e.g.,Meena, R.; Chhatbar, M.; Prasad, K.; Siddhanta, A. K. Development of arobust hydrogel system based on agar and sodium alginate blend. Polym.Int. 2008, 57, 329-336). Agar and sodium alginate were dissolvedseparately in distilled water. Agar was dissolved using microwaveirradiation at 90° C. for 2 min, while the sodium alginate was dissolvedat ambient temperature. Blends of agar and sodium alginate were preparedby mixing agar them in different ratios. The resulting blends werecooled, forming gels which were cut into small pieces and dehydratedwith isopropanol. Dewatered, hardened gel particles were filteredthrough a nylon cloth under reduced pressure and dried in air, followedby drying in an oven at 50° C. for 2 h. The dried blend samples wereground using a mortar and pestle to obtain 20-40 mesh particles.

Hydrogels consisting of a gellan co-crosslinked with scleroglucan havealso been reported (see, e.g., Alhaique, F.; Coviello, T.; Rambone, G.;Carafa, M.; Murtas, E.; Riccieri, F. M.; Dentini, M.; Desideri, P. Agellan-scleroglucan co-crosslinked hydrogel for controlled drugdelivery. Proceedings of the International Symposium on ControlledRelease of Bioactive Materials 1998, 25^(th) 866-867). The use of bothgellan and scleroglucan in the hydrogel resulting in better stiffnessand stability of the macroscopic hydrogel, and resulted in a slowerrelease of the guest molecule. The release rate was undesirably highfrom the Ca²⁺ crosslinked gellan alone.

Kim et al. have synthesized polysaccharide-based hydrogel usingphotocrosslinking of modified dextran (see, e.g., Kim, S. H.; Won, C.Y.; Chu, C. C. Synthesis and characterization of dextran-based hydrogelprepared by photocrosslinking Carbohydrate Polymers 1999, 40, 183-190).Dextran contains (1→6)-linked α-D-glucopyranosyl residues, and threehydroxyl groups per glucose residue in the structure. Dextran was firstreacted with bromoacetyl bromide. The bormoacetylated dextran was thenreacted with sodium acrylate for incorporating vinyl groups.Photocrosslinking was achieved by irradiating the acrylated dextran byUV light. These were macroscopic gels, and not micro- or nanoparticles.

Similarly, Reis et al. introduced pendent vinyl groups by reactingstarch with glycidyl methacrylate (see, e.g., Reis, A. V.; Guilherme, M.R.; Moia, T. A.; Mattoso, L. H. C.; Muniz, E. C.; Tambourgi, E. B.Synthesis and characterization of starch-modified hydrogel as potentialcarrier for drug delivery system. J. Polym. Sci.: Part A: Polym. Chem.2008, 46, 2567-2574). Starch is a polysaccharide composed of twostructural components: amylase and amylopectin. Amylase is a linearchain consisting of 250-300 (1→4)-linked α-D-glucose residues.Amylopectin is a branched molecule consisting of about 1400 D-glucoseresidues with α(1→4) and α(1→6) linkages. It constitutes about 80% ofthe total starch and can be easily hydrolyzed. Crosslinkable vinylgroups were introduced by the reaction of starch with glycidylmethacrylate resulting in structures schematically shown in FIG. 7.

Chen et al. synthesized polymerizable saccharide monomers, such assucrose, by reaction of the sugar with epoxy acrylate, or methacryloylchloride and acetyl chloride, and used these monomers to form hydrogels(see, e.g., Chen, J.; Bongjo, S.; Park, K. Hydrophilic, hydrophobic, andthermoreversible saccharide gels and foams, and methods for producingsame. U.S. Pat. No. 6,018,033, Jan. 25, 2000). Cai et al. have similarlymodified hydropropylcellulose by covalently attaching vinyl groups thatallowed chemical linking of the polysaccharide chains into nanoparticlesthrough a free radical polymerization process (see, e.g., Cai, T.; Hu,Z.; Ponder, B.; St. John, J.; Moro, D. Synthesis and study of andcontrolled release from nanoparticles and their networks based onfunctionalized hydroxypropylcellulose. Macromolecules 2003, 36,6559-6564).

As mentioned previously, reports on encapsulation of hydrophilicmolecules in particulate carriers of aqueous dispersions are notnumerous. Edlund and Albertsson varied the crosslink density ofhemicellulose-based hydrogel microspheres and found that the crosslinkednetwork was not able to retard the rapid release of the small andhydrophilic molecules of compounds such caffeine (see, e.g., Edlund, U.;Albertsson, A.-C. A microspheric system: hemicellulose-based hydrogels.Journal of Bioactive and Biocompatible Polymers 2008, 23, 171-186). Onthe contrary, release of a macromolecule such as protein (bovine serumalbumin) could be controlled by varying the network mesh size, with therelease proceeded by Fickian diffusion. Other studies have shown thesame dependence of size and hydrophilicity of entrapped molecules.

Applicants synthesized hydroxypropyl cellulose microgels usingrelatively non-toxic crosslinking agents such as trisodiumtrimetaphosphate (TSTMP) and sodium tripolyphosphate (STPP).Hydroxypropyl cellulose (HPC) is prepared by base-catalyzed reaction ofpropylene oxide with cellulose. HPC is permitted in foods for humanconsumption, and is described under section 121.1160 of the U.S. Foodand Drug Administration regulations [Klug, E. D. HydroxypropylCellulose. In Encyclopedia of Polymer Science and Technology; Bikales,N. M., Ed.; Wiley Interscience: New York, 1971; Vol. 15, pp 307-314]. Upto 0.4 wt % of unreacted TSTMP and STPP are permissible in food productsaccording to FDA regulations. Other reagents permitted by FDA for makingfood grade starch, such as phosphoryl chloride, adipate, andadipic-acetic mixed anhydride, may also be used for the crosslinkingreaction. Carcinogens such as epichlorohydrin, although used in the pastfor crosslinking starch, can obviously not be used. See, e.g., FIG. 10which depicts crosslinking of hydroxyl containing polysaccharides usingTSTMP in the presence of sodium hydroxide.

Crosslinking of starch using trisodium trimetaphosphate has beentypically carried out in aqueous media at pH of 11.5 [Xie, S. X.; Liu,Q.; Cui, S. W. Starch modification and application. In FoodCarbohydrates: Chemistry, Physical Properties, and Applications; Cui, S.W. Ed.; Taylor & Francis: New York 2005; p. 358]. The reaction isallowed to proceed at 40° C. for 2 to 6 h. The applicants found thathydroxypropyl cellulose microparticles could be obtained, at relativelyhigh concentrations (up to 10 wt %, without macrophase separation),using significantly higher sodium hydroxide concentration and reactiontemperature. Sodium hydroxide not only participates in the crosslinkingreaction, but also, evidently, lowers the LCST of hydroxypropylcellulose resulting in particle formation even at room temperature (atsufficiently high concentrations of NaOH).

Hydroxypropyl cellulose powder, obtained from Sigma-Aldrich, was usedfor microparticle synthesis. The HPC polymer had a number-averagemolecular weight, M _(n), of 10,000 g/mol, a weight-average molecularweight, M _(w), of 80,000 g/mol, a degree of substitution, DS, of 2.5,and a molar substitution, MS, of 3.7. The degree of substitution, DS, isdefined as the average number of hydroxyl groups substituted peranhydroglucose unit [Klug, E. D. Hydroxypropyl Cellulose. InEncyclopedia of Polymer Science and Technology; Bikales, N. M., Ed.;Wiley Interscience: New York, 1971; Vol. 15, pp 307-314]. The molarsubstitution, MS, is defined as the average number of propylene oxidemolecules combined per anhydroglucose unit.

About 15 mg of refined soy lecithin (MP Biomedicals) was dissolved in 5mL of a sodium hydroxide solution (pH=12) to obtain a pale yellowtranslucent solution. Four hundred milligram of HPC was added to thissolution and stirred to result in a viscous solution. In another vial, a12% (w/v) solution of TSTMP was prepared in distilled water. Fivemilliliters of this TSTMP solution was then added to the HPC/soylecithin solution. The mixture was stirred to obtain a homogeneoussolution, which was heated at 50° C. for 1 h and subsequently cooled toroom temperature. The pH of the resulting dispersion, measured using astainless steel ISFET pH probe (IQ Scientific Instrument), was 7.8. ThepH was adjusted to 7 using a few microliters of 4 M hydrochloric acid.The HPC dispersion consisted of: 400 mg of HPC (3.2 mmol of hydroxylgroups), 15 mg (0.05 mmol) soy lecithin, 600 mg (2.0 mmol) of TSTMP, andabout 12 mg (0.3 mmol) sodium hydroxide in about 10 mL of distilledwater. The number-average particle diameter was 3.5 μm and theweight-average particle diameter was 3.7 μm. The viscosity of thedispersion was about 11 cP. Ten milliliters of a 20% (w/v) dextrosesolution in distilled water was then added to this dispersion, and themixture was heated at 60° C. for 10 min. The number-average particlediameter remained nearly the same (˜5 μm) after addition of dextrose.The viscosity of the final dispersion was about 5 cP. The averagediameter of the particles in the dispersion was determined using aALVS-NIBS High Performance Particle Sizer (ALV-GmbH, Langen/Germany).Dispersion viscosity was determined using a Ubbelohde Viscometer (CannonInstrument Co., Pennsylvania).

There were no significant differences in the particle sizes or thedispersion viscosities when the formulations were heated at 50° C. for 3h instead of 1 h.

In another formulation, 10 mL of a 4% (w/v) solution of HPC in distilledwater was taken in a glass vial. Sodium hydroxide pellets (310 mg, 7.75mmol) were added and dissolved in to this solution. The addition ofsodium hydroxide resulted in a cloudy homogeneous dispersion. TSTMP (600mg, 1.96 mmol) and soy lecithin (14 mg, 0.043 mmol) were subsequentlyadded and dissolved. The dispersion was heated at 50° C. for 1 h, afterwhich it was cooled to room temperature. The procedure resulted in theformation of macroparticles that settled to the bottom of the vial.Immediately after cooling, the dispersion was stirred (using a magneticstirrer) and neutralized to pH 7 using 4 M hydrochloric acid. Thenumber- and weight-average particle diameters in the supernatant phasewere about 610 nm and 690 nm, respectively. The viscosity of the HPCdispersion was about 1.6 cP. Ten milliliters of a 20% (w/v) dextrosesolution in distilled water was then added to this dispersion, and themixture was heated at 60° C. for 10 min. The number-average particlediameter in the dextrose loaded dispersion was about 1.6 μm and theweight-average particle diameter was about 2.2 μm after addition ofdextrose. The viscosity of the final dispersion was about 2 cP.

In another embodiment, heating a solution of 4 g of HPC (31.9 mmol ofhydroxyl groups) in 100 g of water with 2.1 g (52.5 mmol) of sodiumhydroxide and 1 g (3.27 mmol) of TSTMP at 110° C. for 2 h, resulted inthe formation of hydrogel microspheres. The dispersion was cooled toroom temperature and neutralized using about 4 mL of 4 M hydrochloricacid to result in a solution with a viscosity of about 22 cP and aweight-average particle diameter of about 3.4 μm. Addition of 104 mL of20% (w/v) dextrose solution gave a final dispersion with a sugarconcentration of 10% (w/v), a viscosity of 6.8 cP and a weight-averageparticle diameter of about 4.1 μm. The formulation was heated at 60° C.for 10 min after the addition of sugar solution.

In another formulation, 8 g of HPC (63.7 mmol of hydroxyl groups)dissolved in 100 g of water was heated with 2.23 g (55.8 mmol) of sodiumhydroxide and 1 g (3.27 mmol) of TSTMP. Heating was carried out in asealed glass reactor at 110° C. for 2 h. After cooling, the unreactedsodium hydroxide was neutralized using about 20 mL of 4 M hydrochloricacid, to yield a dispersion of crosslinked HPC microspheres with aweight-average particle diameter of about 4.3 μm. The viscosity of thedispersion was about 31.2 cP. A 20% (w/v) dextrose solution (120 mL) wasthen added to obtain a formulation with 10% (w/v) dextrose, 3.3% (w/v)HPC, about 2.5% (w/v) sodium chloride. The dispersion was heated at 60°C. for 10 min after sugar addition. The weight-average particle diameterin the final dispersion was about 4.5 μm, and the dispersion viscositywas about 31 cP. The dispersion viscosity was sensitive to the order inwhich the solutions were mixed. If the dextrose solution was added afterthe second heating step (60° C. for 10 min), the viscosity of theresulting dispersion was higher (about 55 cP).

Microparticle hydrogels of hydroxypropyl cellulose and sodium alginate(CAS no. 9005-38-3; American International Chemical, Inc., F-200) aresynthesized as follows. Ten milligrams of HPC (0.080 mmol of hydroxylgroups) was dissolved in 1 mL of distilled water. To this solution wasadded 1 mL of 2.5 M NaOH solution (2.5 mmol NaOH), 20 mg (0.065 mmol) oftrisodium trimetaphosphate, 10 mg of sodium alginate and 2 mg (6.1 μmol)of soy lecithin. The solution was stirred thoroughly. A cloudydispersion was obtained that remained stable even after adding a fewdrops of concentrated hydrochloric acid (leading to a final pH of about2, simulating the acidic environment of the stomach).

Hydroxypropyl cellulose self-assembles in water at a temperature greaterthan 41° C. This temperature, above which spontaneous self-assembly ofthe polymer chain occurs, is called the lower critical solutiontemperature (LCST). Thermal self-assembly of HPC is a reversibleprocess. Individual polymer chains constituting the microparticles getsolvated by water molecules when the solution is cooled below the LCST.Crosslinking the HPC chains using trisodium metaphosphate (TSTMP)prevents dissolution of the microparticles when the solutions are cooledbelow the critical solution temperatures.

In another strategy, crosslinking may be achieved by functionalizing thepolysaccharide using acryloyl (or methacryloyl) groups using acryloylchloride (or methacryloyl chloride). Formation of acryloyl estersresults from the reaction of acryloyl chloride with the hydroxyl groupsof the polysaccharide (FIG. 11). It is important, however, to completelyremove unreacted acryloyl chloride from the functionalized polymer,because of toxicity of acryloyl chloride. The vinyl functionalized HPCmay then be crosslinked in water, above the LCST, using a relativelybenign free-radical redox-initiator such as ascorbic acid and hydrogenperoxide, or thermal initiator such as potassium persulfate.

Thus, 1 g of hydroxypropyl cellulose (8 mmol) was taken in a roundbottom flask equipped with a magnetic stir bar and fitted with a rubberseptum. The polymer was dissolved in 20 mL of anhydrous dichloromethaneto obtain a cloudy, viscous solution. The air in the flask was purgedwith dry nitrogen. About 1 mL (7 mmol) of triethyl amine was injected into the reactor, followed by drop-wise addition of about 520 μL (6.4mmol) of acryloyl chloride. The mixture was stirred at room temperature,whereupon the cloudy solution became clear few minutes after theaddition of acryloyl chloride. The solution was stirred overnight, afterwhich the acrylated hydroxypropyl cellulose product was recovered andpurified by repeated precipitations in cold (˜0° C.) diethyl ether andacetone. The product was dried in vacuo at 40° C. About 40 mg of theacrylated HPC polymer was dissolved in 2 mL distilled water to obtain acloudy solution at room temperature. About 65 mg (200 mmol) of soylecithin was added to this solution and dissolved. The solution of HPCand soy lecithin was de-oxygenated by bubbling nitrogen gas, after whicha 2 mL of a degassed solution of ammonium persulfate (9.1 mg, 40 mmol)was injected. The solution was heated at 70° C. for 2 h to obtain adispersion of crosslinked acrylated hydroxypropyl cellulose particles.The number-average and weight-average particle diameters were 1.28 μmand 1.34 μm, respectively.

In an emulsion-based synthesis of hydroxypropyl cellulose microgels, 80mg of acrylated hydroxypropyl cellulose was dissolved in 2 mL ofdichloromethane. Distilled water (4 mL) was added to this solution andstirred to obtain an emulsion. Crosslinking of the acrylatedhydroxypropyl cellulose was carried out at 35° C. using a redox systemof ammonium persulfate and dextrose. Dextrose (21.6 mg, 12 mmol) wasdissolved in the emulsion. Two milliliters of a solution of ammoniumpersulfate (27.4 mg, 0.12 mmol) in distilled water (2 mL) was injectedin to the emulsion to initiate the crosslinking reaction.Dichloromethane was removed from the resulting dispersion using a rotaryevaporator. A cloudy dispersion of crosslinked acrylated hydroxypropylcellulose microgels was obtained. The crosslinked particles settled tothe bottom of the vial on standing, and could therefore be isolated in apowder form by decanting the supernatant. The crosslinking may also becarried out using redox systems such as persulfate/glucose, hydrogenperoxide/ascorbic acid, etc.

Scanning electron microscopy of a 400 mg HPC, 100 mg TSTMP, 200 mg NaOH,10 mL water solution heated at 110° C. for 2 h, wherein the dispersionwas neutralized with concentrated HCl acid revealed large (˜1 μm) cubicparticles seen under SEM. HPC has a low glass transition temperature andreadily forms a film on the SEM substrate at room temperature. However,it was difficult to image the nanoparticles using SEM.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

What is claimed is:
 1. A composition for in vivo consumption comprising:nutritional supplements; and, compounds for time controlled andsustained release of the nutritional supplements in vivo.
 2. Thecomposition of claim 1, wherein the nutritional supplements comprisecarbohydrates, amino acids, lipids, electrolytes and vitamins.
 3. Thecomposition of claim 1, wherein the compounds for the time controlledand sustained release of the nutritional supplements comprisebiodegradable polymers, bioadhesives and/or binders.
 4. The compositionof claim 3, wherein the biodegradable polymers and binders comprisepoly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetyls,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylate)s, copolymers of polyethylene glycol and polyorthoester,biodegradable polyurethanes, hydrogels, blends and copolymers thereof.5. The composition of claim 1, wherein the nutritional supplements arein a concentration and released in vivo, in a time controlled andsustained release to increase exercise duration and cumulative poweroutput potential by at least about 1% to about 50%, about 50% to about100%, about 100% to about 500% or about 500% to about 1000% whencompared to a control with no nutritional supplement ingestion pre andduring exercise.
 6. The composition of claim 1, wherein the nutritionalsupplements are in a concentration and released in vivo, in a timecontrolled and sustained release to increase burst energy duration andpower output by at least about 1% to about 50%, about 50% to about 100%,about 100% to about 500% or about 500% to about 1000% when compared to acontrol with no nutritional supplement ingestion pre and duringexercise.
 7. The composition of claim 1, wherein the nutritionalsupplements are in a concentration and released in vivo, in a timecontrolled and sustained release to increase the number of effectiveburst events by at least about 1% to about 50%, about 50% to about 100%,about 100% to about 500% or about 500% to about 1000% when compared to acontrol with no nutritional supplement ingestion pre and duringexercise.
 8. The composition of claim 1, wherein the nutritionalsupplements are in a concentration and released in vivo, in a timecontrolled and sustained release to decrease the duration of recoverytime between burst events by at least about 1% to about 50%, about 50%to about 100%, about 100% to about 500% or about 500% to about 1000%when compared to a control with no nutritional supplement ingestion preand during exercise.
 9. The composition of claim 1, wherein thenutritional supplements are in a concentration and released in vivo, ina time controlled and sustained release increase continuous exercisepower output (watts) by about 1% to about 50%, about 50% to about 100%,about 100% to about 500% or about 500% to about 1000%, advantageouslyabout 1% to about 50% at about VO₂ 62% and about 1% to about 25%increase in high intensity (Burst) exercise at about VO₂ 86%, andincrease the number of effective “burst” periods by about 1% to about50%, about 50% to about 100%, about 100% to about 500% or about 500% toabout 1000% as compared to performance when utilizing commerciallyavailable performance enhancing drinks.
 10. The composition of claim 1,wherein the compounds for time controlled and sustained release of thenutritional supplements in vivo release the nutritional supplements ineffective amounts and concentrations over sustained periods of time toincrease exercise duration and cumulative power output potential by atleast a about 1% to about 50%, about 50% to about 100%, about 100% toabout 500% or about 500% to about 1000%, increase burst energy durationand power output by at least about 1% to about 50%, about 50% to about100%, about 100% to about 500% or about 500% to about 1000%, increasethe number of effective burst events from about 1% to about 50%, about50% to about 100%, about 100% to about 500% or about 500% to about1000%, decreases the duration of recovery time between burst events byat least about 1% to about 50%, about 50% to about 100%, about 100% toabout 500% or about 500% to about 1000%, when compared to a control withno nutritional supplement ingestion pre and during exercise.
 11. Thecomposition of claim 1, wherein the compounds for time controlled andsustained release of the nutritional supplements in vivo release thenutritional supplements in effective amounts and concentrations oversustained periods of time to increase continuous exercise power output(watts) by about 1% to about 50%, about 50% to about 100%, about 100% toabout 500% or about 500% to about 1000%, advantageously about 1% toabout 50% at about VO₂ 62% and about 1% to about 25% increase in highintensity (Burst) exercise at about VO₂ 86%, and increase the number ofeffective burst periods by at least about 100% as compared toperformance when utilizing commercially available performance enhancingdrinks.
 12. The composition of claim 1, wherein the composition is abeverage, a gum, a food snack or a powder.
 13. A method of improvingathletic performance and endurance comprising administering to anindividual a composition comprising nutritional supplements and,compounds for sustained release of the nutritional supplements in vivo,wherein the nutritional supplements comprise carbohydrates, amino acids,and electrolytes; and, consuming the composition wherein the compoundsfor sustained release of the nutritional supplements in vivo release thenutritional supplements in effective amounts and concentrations overextended periods of time to increase exercise duration and cumulativepower output potential by at least about 1% to about 50%, about 50% toabout 100%, about 100% to about 500% or about 500% to about 1000%,increase burst energy duration and power output by at least about 1% toabout 50%, about 50% to about 100%, about 100% to about 500% or about500% to about 1000%, increase the number of effective burst events fromabout 1% to about 50%, about 50% to about 100%, about 100% to about 500%or about 500% to about 1000%, decreases the duration of recovery timebetween burst events by at least about 1% to about 50%, about 50% toabout 100%, about 100% to about 500% or about 500% to about 1000%, whencompared to a control with no nutritional supplement ingestion pre andduring exercise; and, improving athletic performance and endurance. 14.The composition of claim 1, wherein the composition comprises ahydrogel.
 15. The composition of claim 14 wherein the hydrogel comprisesa modified polysaccharide.
 16. The composition of claim 15 wherein themodification is selected from the group consisting of carboxylic acid,acryloyl or methacryloyl, hydroxyalkyl, ester, grafted fatty acidmoieties, oligo(alkylene oxide) grafts, functionalized hydrophobes suchas cholesterol, grafted amphiphiles such as alkenylsuccinate, or sidechain derivatizations to introduce aldehydic or carboxylic groups. 17.The composition of claim 14 wherein the hydrogels increase the retentiontime of nutrients in the small intestine because of the mucoadhesivenature of the hydrogel particles.
 18. The composition of claim 14wherein the hydrogels are formed by crosslinking.
 19. The composition ofclaim 18 wherein the crosslinking is performed using free radicalinitiators such as persulfate salts, or redox systems involving ascorbicacid, or a naturally occurring crosslinker such as genipin.
 20. Thecomposition of claim 19 wherein the crosslinking is ionic crosslinking,preferably wherein the ionic crosslinking is with an anionicpolysaccharide such as gellan.